Methods for determining properties that affect an expression property value of polynucleotides in an expression system

ABSTRACT

A method of determining a property that affects expression of polynucleotides in an expression system. A plurality of polynucleotides, each encoding a polypeptide sequence, is constructed. An amino acid is encoded a plurality of times in both a first and second polynucleotide in the plurality. The amino acid is encodable by a plurality of synonymous codons including a first codon. The first codon is in the first polynucleotide at a first frequency relative to other synonymous codons, and is in the second polynucleotide at a second frequency relative to other synonymous codons. The first and second frequencies are different. Each polynucleotide is individually expressed in the expression system to measure an expression property value of the polynucleotides, thereby determining a property that affects expression of polynucleotides. The property is an effect that a frequency of use of one or more codons has on expression of polynucleotides in the expression system.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

The research described in this application was funded in part by NSFSBIR grant 0638333.

1. FIELD OF THE INVENTION

This invention relates to methods for designing nucleic acids withdesired properties, particularly for the expression of encoded proteins.

2. BACKGROUND OF THE INVENTION

It is frequently desirable to express proteins encoded by nucleic acids,for example for production of the protein to be used in a therapeutic orbiocatalytic application, or for the protein to perform a functionwithin the cell in which it is expressed. Due to the degeneracy of thegenetic code, there are numerous different nucleotide sequences that canall encode the same protein. Redesigning a naturally occurring genesequence by choosing different codons without necessarily altering theencoded amino acid sequence often dramatically increases proteinexpression levels (Gustafsson et al, 2004, “Codon bias and heterologousprotein expression,” Trends Biotechnol 22, 346-53).

The inspiration for most codon optimization algorithms comes fromassessing coding sequence characteristics present in naturally derivedgenomic sequences as a proxy for synthetic genes. The assumption guidingthis method is that synthetic genes will express well if the genesequence mimic the nucleotide sequence characteristics of the hostgenome. Variables such as codon adaptation index (CAI), mRNA secondarystructures, cis-regulatory sequences, GC content and many other similarvariables have been shown to somewhat correlate with protein expressionlevels (Villalobos et al, 2006, “Gene Designer: a synthetic biology toolfor constructing artificial DNA segments,” BMC Bioinformatics 7, 285). Aproblem with these correlations is that protein expression is generallybelieved to be controlled at the level of initiation of transcriptionand translation, not translational velocity. These factors arecontrolled by promoter strength and the strength of the ribosome bindingsite, which are different for every natural protein, and which are nottaken into account in such blunt analyses as the most common codon for aparticular amino acid in every protein in an organism's genome. Thesequence characteristics of the coding sequences may reflect otherfactors such as evolutionary constraints involved in facilitating DNAreplication, mutational bias, intrinsic metabolic regulation, transposonresistance, ancestral origin etc. rather than serving as a useful guideto design principles with which to obtain high levels expression ofrecombinant protein (Moura et al, 2005, “Comparative context analysis ofcodon pairs on an ORFeome scale,” Genome Biol. 6, R28).

To date, there has been no systematic study of the effect of codonchoices on protein expression, while keeping other expression controlelements, such as promoters and ribosome binding sequences, constant.Thus there is currently no reliable strategy for selecting the codons ina synthetic gene to obtain high protein expression levels, nor is therecurrently a reliable algorithm with which to assess the likely level ofprotein expressed from a synthetic gene. There is thus a need in the artfor both of these.

3. SUMMARY OF THE INVENTION

The systems and methods described here apply computational biology anddata mining techniques to important molecular design problems. Inparticular, novel ways to map codon sequence space for polynucleotidesequences that encode polypeptides are described. Such maps are used todirect modifications of polynucleotide sequences in order to obtaindesired expression characteristics of encoded polypeptides.

Methods are disclosed for biological engineering using the design andsynthesis of plurality of polynucleotides containing designed codonsubstitutions that are representative of a codon space (the “codonvariant set”). The codon space for a polypeptide is defined by thesequence of that polypeptide. The set of all polynucleotides that encodea single defined polypeptide sequence define the codon space for thatpolypeptide sequence. Because there are sometimes slight variations inthe genetic code in different organisms, the codon space for apolypeptide sequence also depends upon the expression system to be used.

The information used to create the codon substitutions that define acodon variant set can be derived from one or more of: (i) variation ofcodon biases between two or more organisms or genes or syntheticallyconstructed bias tables, (ii) variation in the degree of codon biaswithin an organism, gene, or set of genes, (iii) systematic variation ofcodons including context, (iv) variation of codons according to theirdecoding tRNAs, (v) variation of codons according to GC %, eitheroverall or in one position of the triplet, (vi) variation in degree ofsimilarity to a reference sequence for example a naturally occurringsequence, (vii) variation in the codon frequency cutoff, (viii)structural properties of mRNAs transcribed from the DNA sequence, (ix)prior knowledge about the function of the DNA sequences upon whichdesign of the codon substitution set is to be based, and/or (x)systematic variation of codon sets for each amino acid. The systematicdesign and synthesis of codon variants is an aspect of the presentinvention.

After synthesis, the plurality of polynucleotides containing designedcodon substitutions is characterized functionally to measure a propertyof interest, for example an expression property. Examples of expressionproperties, include but are not limited to, expression of a polypeptide,expression of a polypeptide in soluble form, or expression of apolypeptide in biologically or chemically active form.

An optional additional step in this method is to model asequence-expression relationship between (i) one or more variables thatare characteristics of the polynucleotide sequences themselves (a“polynucleotide sequence property”, including but not limited to thecodon bias, the relative or absolute frequency with which specificsequence elements, including but not limited to codons, are used, GCcontent of the polynucleotide, predicted mRNA secondary structure) and(ii) the property measured for all or the portion of the variants in thevariant set (an “expression property” including but not limited to theexpression of polypeptide by translation of the polynucleotide in anexpression system, the expression of soluble polypeptide by translationof the polynucleotide in an expression system or the expression ofbiologically or chemically active polypeptide by translation of thepolynucleotide in an expression system). The codon variant set may thenoptionally be redefined to comprise new variants that are designed tohave modified values for one or more polynucleotide sequence properties,which values are selected based on a function of the modeledsequence-expression relationship.

In some embodiments, the method further comprises repeating themeasuring, modeling, and, optionally, the redefining, until a variant inthe codon variant set exhibits a value for the expression property thatexceeds a predetermined value.

In some embodiments, the method further comprises repeating themeasuring, modeling, and, optionally, the redefining, until a variant inthe codon variant set exhibits a value for the expression property thatis less than a predetermined value.

In some embodiments, the method further comprises repeating themeasuring, modeling, and, optionally, the redefining, a predeterminednumber of times (e.g., two, three, four, or five times).

In some embodiments, the modeled sequence-expression relationshipcomprises a plurality of values and each value in the plurality ofvalues describes a relationship between (i) a polynucleotide sequenceproperty and an expression property, (ii) a plurality of polynucleotidesequence properties and an expression property, or (iii) a plurality ofpolynucleotide sequence properties and a plurality of expressionproperties.

In some embodiments, the modeling comprises linear regression,non-linear regression, logistic regression, multivariate data analysis,or partial least squares projection to latent variables.

In some embodiments, the modeling comprises computation of a neuralnetwork, computation of a Bayesian model, a generalized additive model,a support vector machine, or classification using a regression tree. Insome embodiments, the modeling comprises boosting or adaptive boosting(See, for example, Hastie, 2003, The Elements of Statistical Learning,Springer, N.Y.).

In some embodiments, the redefining further comprises (i) computing apredicted score for a population of variants of the polynucleotide ofinterest using the modeled sequence-expression relationship, where eachvariant in the population of variants includes a codon substitution atone or more codons in the polynucleotide of interest and (ii) selectingthe codon variant set from among the population of variants as afunction of the predicted score received by each variant in the set ofcodon variants.

In some embodiments, the modeling further comprises modeling a pluralityof relationships between one or more polynucleotide sequence propertiesand one or more expression properties. Each respective relationship inthe plurality of relationships describes the relationship between (i)one or more polynucleotide sequence properties and (ii) one or moreexpression properties measured for all or the portion of the variants inthe codon variant set. Furthermore, the step of redefining the codonvariant set (e) comprises redefining the codon variant set to comprisevariants that include polynucleotide sequence properties that areselected based on a combination of the plurality of sequence-expressionrelationships.

In some embodiments, the codon variant set consists of between 5 and 200variants of the polynucleotide of interest or between 15 and 50 variantsof the polynucleotide of interest.

In some embodiments of the invention the expression property is measuredin one of the following expression systems: bacterial expression systemsincluding Escherichia coli, Salmonella species, Bacillus species,Streptomyces species, Pseudomonas species, Ralstonia eutropha,Chlamydomonas species; yeast expression systems including Saccharomyces,Pichia, Klebsiella and Candida species, Saccaromyces cerevisiae, Pichiapastoris, Pichia methanolica, Klebsiella lactis; fungal expressionsystems including Cryptosporidium and Trichoderma species, filamentousfungal protein production systems, protozoan expression systemsincluding Plasmodium falciparum (the causative agent of malaria),Leishmania model organisms including Caenorhabditis elegans, Drosophilamelanogaster, Xenopus laevis; plants including soybean, bushbean, maize,cotton, tobacco, Arabidopsis, tissue culture expression systemsincluding COS cells, Chinese Hamster Ovary cells and fibroblastsincluding 3T3 cells, cell lines infected with adenovirus, insect celllines such as those derived from Spodptera species for growingbaculovirus; model organisms for the study of disease and tests of theefficacies of DNA vaccines such as macaques, mice, rats, guinea pigs,sheep, goats and rabbits; in vitro expression systems prepared fromextracts of living cells including E. coli extracts, wheat germextracts, rabbit reticulocyte lysates; in vitro expression systemsprepared by assembly of purified individual components.

In some embodiments, the sequence-expression relationship has the form:Y=f(w ₁ x ₁ , w ₂ x ₂ , . . . w _(i) x _(i))

where,

Y is a quantitative measure of the expression property;

x_(i) is a descriptor of a polynucleotide sequence property;

w_(i) is a weight applied to descriptor x_(i); and

f( ) is a mathematical function.

In some embodiments, the modeling comprises regressing:Y=f(w ₁ x ₁ , w ₂ x ₂ , . . . w _(i) x _(i)).

In some instances this regressing comprises linear regression,non-linear regression, logistic regressing, or partial least squaresprojection to latent variables.

A sequence-expression relationship derived from the expressionproperties of a codon variant set of polynucleotides encoding onepolypeptide may be used to design polynucleotides to encode a secondpolypeptide with a different amino acid sequence. The use of asequence-expression relationship to design polynucleotides for theexpression of a polypeptide of interest, where the sequence-expressionrelationship was derived from polynucleotides encoding polypeptides thatare not the polypeptide of interest is an aspect of the invention.

In some embodiments of the invention the modeled sequence-expressionrelationship is used to elucidate design principles for expression ofpolynucleotides in specific expression systems.

4. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates UNAFold-calculated phi29 polymerase gene mRNAstructures. Examples of mRNA structures possible in a 50 nucleotide mRNAwindow at position 147 (1A) or in the 5′ leader (1B) are shown. TheShine-Dalgarno (SD) sequence and the codon for serine at position 10 areindicated. Lowercase letters indicate untranslated nucleotides.

FIG. 2 illustrates the gene design parameter matrix for phi29 polymerasetest variants. Columns are as follows: Codon bias table indicateswhether the codon bias for all E. coli. genes (E. coli.) or E. coli.class II genes (Henaut and Danchin, 1996, “Analysis and predictions fromEscherichia coli sequences,” Escherichia Coli and SalmonellaTyphimurium: Cellular and Molecular Biology, 2047-2066) (E. coli. II)was used. Threshold indicates the codon frequency a codon has to exceedin order to be used in the design. CAI is the codon adaptation index,which was not used directly as a design parameter, but was monitored.RNA@147 is the value for the predicted lowest free energy mRNA secondarystructure for nucleotides 147-196 of the coding sequence. 5′RNA is thevalue for the predicted lowest free energy mRNA secondary structure fornucleotides the first 121 nucleotides of the mRNA. 5′AT wobble is thefraction of the first 15 codons using A or T in the third position. GC6and GC7 refer to the number of runs of consecutive G and/or Cnucleotides of length at least 6 or 7, respectively. WTID is thefraction sequence identity to the wild-type sequence. Expression is themean and standard deviation of expression relative to the referenceclone, multiplied by 100.

FIG. 3 illustrates the expression of five phi29 polymerase variants inE. coli strain BL21(DE3) pLysS. Triplicate expression analyses for theindicated variants are shown. The arrow indicates the position offull-length phi29 polymerase. M, molecular weight standards. C,non-induced control expression. “Ref.” indicates replicates of thereference variant. Numbers indicate amount in ng for BSA standards.

FIG. 4 illustrates results from PLS fitting of phi29 polymerase variantexpression data. All variants listed in FIG. 2 except the wild-type geneare included. Nine variables from FIG. 2 shown to contributesignificantly to the model are shown. (Part A) Plot of measuredexpression levels versus those predicted by the PLS model using 2 latentvariables. (Part B) Loading of the variables for LV1, which captures 65%of the expression variance. RNA structure variables are included as theabsolute value of the folding free energies listed in FIG. 2. Thus apositive loading indicates preference for stronger structure.

FIG. 5 shows the encoding of variables for sequence-expression modelingwhen the variables are the possible codons used at the polynucleotidesequence corresponding to each amino acid position. Variant numbers aregiven in the left hand column. The top row lists the variable number,the amino acid position and then codon. For example the second column isheaded “1: 2-AAG”, meaning variable 1 is whether the polynucleotidevariant uses the codon AAG to encode the second amino acid of thepolypeptide. Variants 1, 2 and 6 use the codon AAG at the secondposition, and therefore have a 1 in this column. The third column,headed “2: 2-AAA” indicates that variable 2 is whether thepolynucleotide variant uses the codon AAA to encode the second aminoacid of the polypeptide. Variants 3, 4, 5, 7 and 8 use the codon AAA atthe second position, and therefore have a 1 in this column.

FIG. 6 shows information from a PLS model correlating the level of phi29protein expression in E. coli with the codons used to encode the proteinat each position in the polypeptide. Part A shows the fit betweenpredicted and measured expression levels for a Partial Least Squares(PLS) model of the expression and sequence of the 21 variants describedin FIG. 2. Part B shows the positions in the polynucleotide sequencethat were identified in this model as possible expression determiningpositions or regions.

FIG. 7 compares the codon usage of phi29 variants 15 (solid bars) and 19(striped bars) with the highly expressed genes E. coli class II bias(white bars) (Henaut and Danchin, 1996, “Analysis and predictions fromEscherichia coli sequences,” Escherichia Coli and SalmonellaTyphimurium: Cellular and Molecular Biology, 2047-2066). The number ofinstances of each codon within the gene is shown. Letters indicate codongroups by amino acid where multiple possible codings exist.

FIG. 8 illustrates PLS modeling of phi29 DNA polymerase expression as afunction of the frequency of each of the 59 variable codons. The graphshows correlation between measured and predicted values.

FIG. 9 illustrates the expression of six ScFv variants in E. coli strainBL21(DE3) pLysS. Triplicate expression analyses for the indicatedvariants are shown. The arrow indicates the position of full-lengthphi29 polymerase. M, molecular weight standards. C, non-induced controlexpression. Numbers indicate amount in ng for BSA standards.

FIG. 10 illustrates results from PLS fitting of scFv variant expressiondata. Six variables that contribute significantly to the model areshown. (10A) Plot of measured expression levels versus those predictedby the PLS model using 2 latent variables. (10B) Loading of thevariables for LV1, which captures 44% of the expression variance. RNAstructure variables are included as the absolute value of the foldingfree energies.

FIG. 11 illustrates a PLS modeling of phi29 DNA polymerase expression asa function of the frequency of 12 codons selected as the most importantusing a genetic algorithm. The graph shows correlation between measuredand predicted values.

FIG. 12 shows PLS modeling of ScFv expression as a function of thefrequency of each of the 59 variable codons. The graph shows correlationbetween measured and predicted values.

FIG. 13 shows PLS modeling of ScFv expression as a function of thefrequency of 12 codons selected as the most important using a geneticalgorithm. The graph shows correlation between measured and predictedvalues.

FIG. 14 shows PLS modeling of protein expression as a function of thefrequency of each of the 59 variable codons for combined data from ScFvand phi29 variants. The graph shows correlation between measured andpredicted values.

FIG. 15 shows a fit between measured and predicted expression levels inaccordance with an embodiment of the present invention.

FIG. 16 shows expression of CenpA genes with altered codon bias. PAGEanalysis of total expressed protein from E. coli strains harboring twodifferent genes for cenpA is shown. The lane labeled “ModA” showsexpression from a gene using codon usage derived from a model forexpression (see text). The lane labeled “Coli-II” shows expression froma gene designed to match the codon bias of 27 naturally highly expressedgenes in E. coli. “Control” shows expression from the same host butlacking a CenpA gene. The arrow indicates the position of thefull-length CenpA protein.

5. DETAILED DESCRIPTION OF THE INVENTION

Before the present invention is described in detail, it is to beunderstood that this invention is not limited to the particularmethodology, devices, solutions or apparatuses described, as suchmethods, devices, solutions or apparatuses can, of course, vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto limit the scope of the present invention.

5.1 DEFINITIONS

Use of the singular forms “a,” “an,” and “the” include plural referencesunless the context clearly dictates otherwise. Thus, for example,reference to “a polynucleotide” includes a plurality of polynucleotides,reference to “a substrate” includes a plurality of such substrates,reference to “a variant” includes a plurality of variants, and the like.

Where a range of values is recited, it is to be understood that eachintervening integer value, and each fraction thereof, between therecited upper and lower limits of that range is also specificallydisclosed, along with each subrange between such values. The upper andlower limits of any range can independently be included in or excludedfrom the range, and each range where either, neither or both limits areincluded is also encompassed within the invention. Where a value beingdiscussed has inherent limits, for example where a component can bepresent at a concentration of from 0 to 100%, or where the pH of anaqueous solution can range from 1 to 14, those inherent limits arespecifically disclosed. Where a value is explicitly recited, it is to beunderstood that values which are about the same quantity or amount asthe recited value are also within the scope of the invention. Where acombination is disclosed, each subcombination of the elements of thatcombination is also specifically disclosed and is within the scope ofthe invention. Conversely, where different elements or groups ofelements are individually disclosed, combinations thereof are alsodisclosed. Where any element of an invention is disclosed as having aplurality of alternatives, examples of that invention in which eachalternative is excluded singly or in any combination with the otheralternatives are also hereby disclosed; more than one element of aninvention can have such exclusions, and all combinations of elementshaving such exclusions are hereby disclosed.

Unless defined otherwise herein, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Singleton et al.,1994, Dictionary of Microbiology and Molecular Biology, 2nd Ed., JohnWiley and Sons, New York, and Hale & Marham, 1991, The Harper CollinsDictionary of Biology, Harper Perennial, New York, provide one of skillwith a general dictionary of many of the terms used in this invention.Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, the preferred methods and materials are described. Unlessotherwise indicated, nucleic acids are written left to right in 5′ to 3′orientation; amino acid sequences are written left to right in amino tocarboxy orientation, respectively. The terms defined immediately beloware more fully defined by reference to the specification as a whole.

The terms “polynucleotide,” “oligonucleotide,” “nucleic acid” and“nucleic acid molecule” and “gene” are used interchangeably herein torefer to a polymeric form of nucleotides of any length, and may compriseribonucleotides, deoxyribonucleotides, analogs thereof, or mixturesthereof. This term refers only to the primary structure of the molecule.Thus, the term includes triple-, double- and single-strandeddeoxyribonucleic acid (“DNA”), as well as triple-, double- andsingle-stranded ribonucleic acid (“RNA”). It also includes modified, forexample by alkylation, and/or by capping, and unmodified forms of thepolynucleotide. More particularly, the terms “polynucleotide,”“oligonucleotide,” “nucleic acid” and “nucleic acid molecule” includepolydeoxyribonucleotides (containing 2-deoxy-D-ribose),polyribonucleotides (containing D-ribose), including tRNA, rRNA, hRNA,siRNA and mRNA, whether spliced or unspliced, any other type ofpolynucleotide which is an N- or C-glycoside of a purine or pyrimidinebase, and other polymers containing nonnucleotidic backbones, forexample, polyamide (e.g., peptide nucleic acids “PNAs”) andpolymorpholino (commercially available from the Anti-Virals, Inc.,Corvallis, Oreg., as Neugene) polymers, and other syntheticsequence-specific nucleic acid polymers providing that the polymerscontain nucleobases in a configuration which allows for base pairing andbase stacking, such as is found in DNA and RNA. There is no intendeddistinction in length between the terms “polynucleotide,”“oligonucleotide,” “nucleic acid” and “nucleic acid molecule,” and theseterms are used interchangeably herein. These terms refer only to theprimary structure of the molecule. Thus, these terms include, forexample, 3′-deoxy-2′, 5′-DNA, oligodeoxyribonucleotide N3′ P5′phosphoramidates, 2′-O-alkyl-substituted RNA, double- andsingle-stranded DNA, as well as double- and single-stranded RNA, andhybrids thereof including for example hybrids between DNA and RNA orbetween PNAs and DNA or RNA, and also include known types ofmodifications, for example, labels, alkylation, “caps,” substitution ofone or more of the nucleotides with an analog, internucleotidemodifications such as, for example, those with uncharged linkages (e.g.,methyl phosphonates, phosphotriesters, phosphoramidates, carbamates,etc.), with negatively charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), and with positively charged linkages (e.g.,aminoalkylphosphoramidates, amino-alkyl-phosphotriesters), thosecontaining pendant moieties, such as, for example, proteins (includingenzymes (e.g. nucleases), toxins, antibodies, signal peptides,poly-L-lysine, etc.), those with intercalators (e.g., acridine,psoralen, etc.), those containing chelates (of, e.g., metals,radioactive metals, boron, oxidative metals, etc.), those containingalkylators, those with modified linkages (e.g., alpha anomeric nucleicacids, etc.), as well as unmodified forms of the polynucleotide oroligonucleotide.

Where the polynucleotides are to be used to express encoded proteins,nucleotides that can perform that function or which can be modified(e.g., reverse transcribed) to perform that function are used. Where thepolynucleotides are to be used in a scheme that requires that acomplementary strand be formed to a given polynucleotide, nucleotidesare used which permit such formation.

It will be appreciated that, as used herein, the terms “nucleoside” and“nucleotide” will include those moieties which contain not only theknown purine and pyrimidine bases, but also other heterocyclic baseswhich have been modified. Such modifications include methylated purinesor pyrimidines, acylated purines or pyrimidines, or other heterocycles.Modified nucleosides or nucleotides can also include modifications onthe sugar moiety, e.g., where one or more of the hydroxyl groups arereplaced with halogen, aliphatic groups, or is functionalized as ethers,amines, or the like.

Standard A-T and G-C base pairs form under conditions which allow theformation of hydrogen bonds between the N3-H and C4-oxy of thymidine andthe N1 and C6-NH₂, respectively, of adenosine and between the C2-oxy, N3and C4-NH₂, of cytidine and the C2-NH₂, N′—H and C6-oxy, respectively,of guanosine. Thus, for example, guanosine(2-amino-6-oxy-9-β-D-ribofuranosyl-purine) may be modified to formisoguanosine (2-oxy-6-amino-9-β-D-ribofuranosyl-purine). Suchmodification results in a nucleoside base which will no longereffectively form a standard base pair with cytosine. However,modification of cytosine (1-β-D-ribofuranosyl-2-oxy-4-amino-pyrimidine)to form isocytosine (1-β-D-ribofuranosyl-2-amino-4-oxy-pyrimidine-)results in a modified nucleotide which will not effectively base pairwith guanosine but will form a base pair with isoguanosine (U.S. Pat.No. 5,681,702 to Collins et al., hereby incorporated by reference in itsentirety). Isocytosine is available from Sigma Chemical Co. (St. Louis,Mo.); isocytidine may be prepared by the method described by Switzer etal. (1993) Biochemistry 32:10489-10496 and references cited therein;2′-deoxy-5-methyl-isocytidine may be prepared by the method of Tor etal., 1993, J. Am. Chem. Soc. 115:4461-4467 and references cited therein;and isoguanine nucleotides may be prepared using the method described bySwitzer et al., 1993, supra, and Mantsch et al., 1993, Biochem.14:5593-5601, or by the method described in U.S. Pat. No. 5,780,610 toCollins et al., each of which is hereby incorporated by reference in itsentirety. Other nonnatural base pairs may be synthesized by the methoddescribed in Piccirilli et al., 1990, Nature 343:33-37, herebyincorporated by reference in it entirety, for the synthesis of2,6-diaminopyrimidine and its complement(1-methylpyrazolo-[4,3]pyrimidine-5,7-(4H,6H)-dione. Other such modifiednucleotidic units which form unique base pairs are known, such as thosedescribed in Leach et al. (1992) J. Am. Chem. Soc. 114:3675-3683 andSwitzer et al., supra.

The phrase “DNA sequence” refers to a contiguous nucleic acid sequence.The sequence can be either single stranded or double stranded, DNA orRNA, but double stranded DNA sequences are preferable. The sequence canbe an oligonucleotide of 6 to 20 nucleotides in length to a full lengthgenomic sequence of thousands or hundreds of thousands of base pairs.

The term “protein” refers to contiguous “amino acids” or amino acid“residues.” Typically, proteins have a function. However, proteins alsoencompass polypeptides and smaller contiguous amino acid sequences thatdo not have a functional activity. The functional proteins of thisinvention include, but are not limited to, esterases, dehydrogenases,hydrolases, oxidoreductases, transferases, lyases, ligases, receptors,receptor ligands, cytokines, antibodies, immunomodulatory molecules,signalling molecules, fluorescent proteins and proteins withinsecticidal or biocidal activities. Useful general classes of enzymesinclude, but are not limited to, proteases, cellulases, lipases,hemicellulases, laccases, amylases, glucoamylases, esterases, lactases,polygalacturonases, galactosidases, ligninases, oxidases, peroxidases,glucose isomerases, nitrilases, hydroxylases, polymerases anddepolymerases. In addition to enzymes, the encoded proteins which can beused in this invention include, but are not limited to, transcriptionfactors, antibodies, receptors, growth factors (any of the PDGFs, EGFs,FGFs, SCF, HGF, TGFs, TNFs, insulin, IGFs, LIFs, oncostatins, and CSFs),immunomodulators, peptide hormones, cytokines, integrins, interleukins,adhesion molecules, thrombomodulatory molecules, protease inhibitors,angiostatins, defensins, cluster of differentiation antigens,interferons, chemokines, antigens including those from infectiousviruses and organisms, oncogene products, thrombopoietin,erythropoietin, tissue plasminogen activator, and any other biologicallyactive protein which is desired for use in a clinical, diagnostic orveterinary setting. All of these proteins are well defined in theliterature and are so defined herein. Also included are deletion mutantsof such proteins, individual domains of such proteins, fusion proteinsmade from such proteins, and mixtures of such proteins; particularlyuseful are those which have increased half-lives and/or increasedactivity.

“Polypeptide” and “protein” are used interchangeably herein and includea molecular chain of amino acids linked through peptide bonds. The termsdo not refer to a specific length of the product. Thus, “peptides,”“oligopeptides,” and “proteins” are included within the definition ofpolypeptide. The terms include polypeptides containing in co- and/orpost-translational modifications of the polypeptide made in vivo or invitro, for example, glycosylations, acetylations, phosphorylations,PEGylations and sulphations. In addition, protein fragments, analogs(including amino acids not encoded by the genetic code, e.g.homocysteine, ornithine, p-acetylphenylalanine, D-amino acids, andcreatine), natural or artificial mutants or variants or combinationsthereof, fusion proteins, derivatized residues (e.g. alkylation of aminegroups, acetylations or esterifications of carboxyl groups) and the likeare included within the meaning of polypeptide.

“Amino acids” or “amino acid residues” may be referred to herein byeither their commonly known three letter symbols or by the one-lettersymbols recommended by the IUPAC-IUB Biochemical NomenclatureCommission. Nucleotides, likewise, may be referred to by their commonlyaccepted single-letter codes.

The term “codon bias” or “relative codon frequency” refers to therelative frequencies of use of codons that encode the same amino acid(“synonymous codons”). The bias may be naturally occurring, for examplethe codon bias in an organism's genome reflects the relative overall useof synonymous codons within all the genes in that organism. The bias mayalso be used in a computational algorithm, where for example it may beused to determine the relative frequency with which different synonymouscodons are selected for use in designing a polynucleotide sequence.Similarly the “relative” frequency of any sequence element used toencode a polypeptide within a polynucleotide is the frequency with whichthat sequence element is used to encode a feature of the polypeptide,divided by the number of occurrences within the polypeptide in a givenreading frame of features that could be encoded by that sequenceelement.

The terms “codon usage table” or “codon bias table” or “codon frequencylookup table” are used interchangeably to describe a table whichcorrelates each codon that may be used to encode a particular aminoacid, with the frequencies with which each codon is used to encode thatamino acid in a specific organism, or within a specified class of geneswithin that organism, or within one or more synthetic polynucleotides. A“hybrid codon usage table” or “hybrid codon bias table” can also beconstructed by combining two or more codon usage tables according to avariety of possible rules, some of which will be enumerated in moredetail elsewhere herein.

The term “absolute codon frequency” refers to the frequency with which acodon appears relative to the total number of codons (e.g. bothsynonymous and non-synonymous codons) within a polynucleotide or set ofpolynucleotides in a given reading frame (e.g., the reading frame thatis used to encode a protein). Similarly the “absolute” frequency of anysequence element used to encode a polypeptide within a polynucleotide isthe frequency with which that sequence element is used to encode afeature (e.g., amino acid, amino acid pair, etc.) of the polypeptide,divided by the number of occurrences within the polypeptide of featuresof the same size as those that could be encoded by that sequenceelement.

The terms “threshold” or “cutoff” are used interchangeably to refer tothe minimum allowable frequency in using a codon frequency lookup table.For example if a threshold or cutoff of 10% is set for an algorithm touse with a sequence element frequency lookup table, then no sequenceelements with a value of less than 10% in that lookup table are acceptedby the algorithm for subsequent polynucleotide design and synthesis.Thresholds may be expressed as percentages (e.g., the percentage of timethat an organism or class of genes within an organism uses a specifiedcodon to encode an amino acid) or as frequencies (0.1 would be thefrequency of codon usage that could also be expressed as 10%).

The term “splice variant” or “splicing variant” refers to the differentpossible RNA products that may be produced by a cell that transcribes asegment of DNA to produce an RNA molecule. These different productsresult from the action of the RNA splicing and transportation machinery,whose specificity of function differs from cell to cell, causingdifferent signals within an RNA sequence to be recognized as introndonor and acceptor sites, and leading to different RNA products.

The term “expression system” refers to any in vivo or in vitrobiological system that is used to produce one or more proteins encodedby a polynucleotide. Examples of expression systems include, but are notlimited to E. coli, baculovirus, mammalian tissue culture, and plantssuch as maize or soybean.

The term “annealing temperature” or “melting temperature” or “transitiontemperature” refers to the temperature at which a pair of nucleic acidsis in a state intermediate between being fully annealed and fullymelted. The term refers to the behavior of a population of nucleicacids: the “annealing temperature” or “melting temperature” or“transition temperature” is the temperature at which 50% of themolecules are annealed and 50% are separate. Annealing temperatures canbe determined experimentally. There are also methods well know in theart for calculating these temperatures.

The terms “codon space” and “codon sequence space” refer to all of thepossible polynucleotide sequences that can be used to encode a specificpolypeptide, by using different codons (nucleotide triplets) to encodeeach amino acid within the polypeptide.

The term “codon substitution” refers to a process of altering apolynucleotide sequence by changing one or more of the codons encodingone or more amino acids within a polypeptide, though without alteringthe sequence of the encoded polypeptide.

The term “codon variant set” refers to a set of polynucleotidesequences, each of which preferably encodes the same polypeptide or lesspreferably a set of highly similar polypeptides sharing at least 99% or98% or 97% or 96% or 95% or 94% or 93% or 92% or 91% or 90% amino acidsequence identity to at least one other polypeptide in the set, butwhich use different codons at one or more positions within the sequence.

The term “polynucleotide sequence property” is used to describe aproperty that is a direct characteristic of a polynucleotide.Polynucleotide sequence properties include but are not limited to codonbias, the frequency with which specific codons are used, the GC contentof all or a portion of the polynucleotide, the predicted secondarystructure of the polynucleotide or its transcription product.

The term “expression property” is used to describe a property of apolynucleotide in an in vivo or in vitro expression system. Expressionproperties include but are not limited to the amount of RNA or proteinproduced and the amount of soluble or otherwise functional proteinproduced.

The term “codon expression relationship” is used to describe arelationship between one or more polynucleotide sequence properties andone or more expression properties.

The term “translation” refers to the process by which a polypeptide issynthesized by a ribosome ‘reading’ the sequence of a polynucleotide.

5.2 FACTORS AFFECTING PROTEIN EXPRESSION

Because of the degeneracy of the genetic code, one polypeptide sequencemay be encoded by many different polynucleotides. Some of thesepolynucleotides will be easier to synthesize in a high fidelity process,while others will be more difficult. When a polynucleotide is beingdesigned and/or synthesized to encode a polypeptide, a polynucleotidesequence may therefore be chosen that facilitates the high fidelitysynthesis of that polynucleotide, in addition to ensuring that thepolynucleotide will possess the desired expression properties. Methodsfor choosing a polynucleotide sequence that fulfills functional as wellas ease-of-synthesis criteria may be accomplished using computerprograms (e.g., software). The methods and the software for performingthe methods are nonlimiting aspects of some embodiments of the presentdisclosure.

Factors that affect protein expression, the expression of solubleprotein or the expression of active protein encoded by thepolynucleotide fall into several classes, all of which can be influencedby the codons chosen to encode the amino acids of the polypeptide. Firstthere are those that affect mRNA levels within the expression system.These include factors affecting the rate of production of mRNA includingthe RNA polymerase type used for transcription, the RNA polymerase levelpresent in the expression system and the transcription promoter sequenceused. The mRNA levels are also affected by the mRNA degradation rate,which are in turn influenced by mRNA destabilizing motifs, RNAserecognition sequences and polyA addition signals. The mRNA levels mayalso be affected by mRNA structures at the translational initiationsite, at the ribosome binding site, at the start codon, around theinitial 10-50 codons or elsewhere within of following the open readingframe, transcriptional termination motifs present before or within theopen reading frame, signals within the transcribed sequence such asthose that direct, alter or modify mRNA splicing and nuclear export.Second there are factors that affect the translational initiation rate.These include the sequence of the ribosome binding site, sequencesupstream of the ribosome binding site, sequences around the start codon(for example Kozak consensus sequences), the presence, relative locationand sequence of internal ribosome entry sites, the sequence and distancebetween the ribosome entry site or the ribosome binding site or the 5′end of the mRNA and the start codon, the mRNA structures at thetranslational initiation site, at the ribosome binding site, at thestart codon, around the initial 10-50 codons, the sequence of theinitial 10-20 codons, the GC bias of the initial 10-20 codons, the codonused at the codon adjacent to the start codon, the sequence of the startcodon (AUG, UUG, or GUG), the ribosome concentration, the growthconditions before induction of expression, the growth conditions duringexpression, the temperature prior to induction of expression and thetemperature during expression. Third there are factors that affect therate of translational elongation. These include the level of chargedtRNAs (Elf et al., 2003, “Selective charging of tRNA isoacceptorsexplains patterns of codon usage,” Science 300, 1718-1722), which dependupon tRNA concentrations, tRNA charging rates and amino acidavailability. They also include the rate of ribosomal tRNA selection(decoding rate) which depends upon the strength of the codon-anticodoninteraction, the preceding codon (P-site codon), the wobble base of thepreceding codon and the wobble base of the codon being read. Fourth arefactors that affect ribosomal fidelity including those that influenceribosomal frameshifts such as homopolymer stretches, G/C islands, A/Tislands and homopolymer stretches near pause sites. Fifth are peptidesthat are hindered in the ribosomal exit channel, which depend in partupon the amino acid sequence of the initial 10-20 amino acids.

Particularly useful methods for designing polynucleotides are those thatintegrate functional constraints such as the selection of codons thatwill express well in one or more chosen host systems, the elimination ofunwanted restriction sites and the inclusion of desired restrictionsites, with synthesis constraints such as the elimination of repeatedsequence elements and the balancing of GC content throughout thesequence.

5.3 DESIGNING POLYNUCLEOTIDES USING FREQUENCY LOOKUP TABLES

Most organisms use the same genetic code, that is, in general the sametriplet of nucleotides (codon) specifies the same amino acid. Differentorganisms use these codons with different frequencies within theirgenes, however. For example different codon biases are found in humans,human viruses such as hepatitis A, hepatitis B, hepatitis C, humanimmunodeficiency virus (HIV), human papilloma virus (HPV), influenza,flaviviruses, lentiviruses, papovaviruses, human pathogens such asMycobacteria, Chlamydomonas, Candida, Plasmodium falciparum (thecausative agent of malaria), Cryptosporidium, Leishmania and otherprotozoa, model organisms such as Tetrahymena, Arabidopsis, Xenopus,Ralstonia, Drosophila, Caenorhabditis elegans, and commonly usedexpression systems such as baculovirus, Escherichia coli, Bacillus,Pseudomonas species, Salmonella species, Streptomyces species,filamentous fungi, mammalian cell lines including COS cells, ChineseHamster Ovary cells and fibroblasts including 3T3 cells, insect celllines such as those derived from Spodptera species, yeasts includingSaccharomyces, Pichia, Klebsiella and Candida species, plants includingmaize, soybean and cotton and model organisms for the study of diseaseand tests of the efficacies of DNA vaccines such as macaques, mice,rats, guinea pigs, sheep, goats and rabbits, in vitro expression systemsprepared from extracts of living cells, in vitro expression systemsprepared by assembly of purified individual components.

In the systems and methods disclosed herein, frequency lookup tables areconstructed to represent the distribution of codons within a set ofsynthetically designed genes that may share one or more properties suchas the level of expressed protein, the level of expressed solubleprotein or the level of expressed active protein. For example, a set ofsynthetically designed and constructed genes may be experimentallytested in an expression host, and the genes may then be categorizedaccording to one or more functional properties. The frequencies withwhich different codons are used to encode each amino acid may becalculated for the synthetic genes that perform the best, for examplethose that express the most protein, or those that express the mostsoluble protein, or those that express the most active protein. Thesefrequencies can then be used directly in the design of polynucleotides,or they may be incorporated into lookup tables that can then be used inthe design of polynucleotides.

In some embodiments, codons that are very rarely used in a specific hostare eliminated from a frequency lookup table. For example Arg is encodedby six possible codons: CGG, CGA, CGT, CGC, AGG and AGA. Of these,codons CGG, CGA, AGA and AGG each occur only about 1% of the time inhighly expressed E. coli genes, while CGT occurs 64% of the time and CGC33% of the time. It may be advantageous to eliminate the four rarelyused codons from the synthetic polynucleotide entirely. In this caseonly CGT and CGC would be used to encode Arg in the syntheticpolynucleotide.

Threshold values for codons may be selected such that a codon thatappears less frequently in a frequency lookup table than that thresholdvalue are not used in a polynucleotide for expression in that host.Threshold values of 0.1 (10%), 0.09 (9%), 0.08 (8%), 0.07 (7%), 0.06(6%), 0.05 (5%) and 0.04 (4%) can all be useful, where such thresholdvalues represent the minimum frequency value for a codon in a frequencylookup table that allows the respective codon to be used in designing asynthetic polynucleotide. Threshold values can be set using a method inwhich codons are selected probabilistically based upon a frequencylookup table, then codons whose frequency is below the threshold arediscarded and another codon is chosen, again probabilistically.Alternatively a codon frequency lookup table may be pre-calculated withthe frequency for a codon that appears below the threshold frequencybeing set to zero so that it is never selected by a probabilisticselection method.

Hybrid frequency lookup tables may be constructed for designing apolynucleotide encoding a polypeptide to be expressed in more than oneexpression system. One method of constructing such hybrid frequencylookup tables is to combine two or more starting frequency lookup tablesfrom one or more organism. In one combination method, a thresholdfrequency is selected and any codons that fall below the threshold areeliminated from all of the starting frequency lookup tables (where thethreshold frequency refers to the minimum frequency value for a codon ina frequency lookup table that allows the respective codon to be used indesigning a synthetic polynucleotide). For the remaining codons thereare several possible methods of processing the frequencies. An averageof the frequency ranges in the starting frequency lookup tables may beobtained. Alternatively the higher of the frequency ranges may beselected for each of the codons. Another possibility is to select thelower of the frequency ranges for each of the codons. In all cases, suchcombined lookup frequency lookup tables should be used in such a waythat the sum of the frequencies for all codons that encode one aminoacid are equal to 1. By avoiding low frequency codons for multipleorganisms, expression in all of those organisms may be improved, therebyincreasing the general usefulness of the synthetic polynucleotide. Asused herein, a frequency range refers to a continuous set of values(e.g., frequency of occurrence of a codon, frequency of occurrence of asequence element) bounded by a maximum percent occurrence and a minimumpercent occurrence. In other words, a frequency range refers to any andall values (e.g., frequency of occurrence of a codon, frequency ofoccurrence of a sequence element) that fall between a maximum percentoccurrence and a minimum percent occurrence, in addition to the maximumpercent occurrence and a minimum percent occurrence.

Because codon biases from conventional studies contain average valuescompiled from information from more than one gene, the codondistribution for any one gene may not precisely match the values foundin the conventional studies, even if the codon distribution in a genewas in part used to calculate the codon biases. Conversely the codonbias within a polynucleotide may not precisely match the codonpreferences from conventional studies, even if the codon biases fromsuch studies were used to design the polynucleotide, for example if thecodon biases from a study were used to guide a probabilistic choice ofcodons to represent each amino acid. For example, in designing apolynucleotide to encode a polypeptide with an E. coli codon bias, eachtime a Tyr is encountered, a selection method or computer program may beused that has a 35 percent chance of selecting TAT and a 65 percentchance of selecting TAC. On average, many polynucleotides designed bysuch a method would contain TAT and TAC in the ratio of 0.35 to 0.65,although any individual polynucleotide may vary from this ratio. Similarmethods may be used to select codons to encode the other amino acidsfrom the polypeptide.

It is possible to make the codon distribution in a polynucleotide veryclosely match the frequencies in a frequency lookup table (whetherderived from natural sequences or from synthetic sequences). One way inwhich this can be done is to select an initial polynucleotide sequence,and then to calculate the codon distribution within this initialpolynucleotide sequence. Codons in the polynucleotide sequence can thenbe changed until the codon distributions within it are as close asdesired to a specified frequency lookup table. There are many ways toachieve this kind of iterative sequence optimization. For example,codons can be selected at random, a change can be proposed at random andthe change can be accepted if it produces a codon distribution that iscloser to the one desired. Codons may also be selected in a directedway. For example, codons in a specific part of the polynucleotide,codons encoding specific amino acids, codons with a specific GC bias,codons with a specified wobble base may be preferentially selected forreplacement or may be preferentially withheld from replacement.

This kind of Monte Carlo algorithm may generally be used to modify anyother quantifiable (including binary or Boolean) polynucleotide sequenceproperty or combination of properties. The inventive steps are (1)select an initial codon sequence to encode a polypeptide; (2) quantifythe one or more polynucleotide sequence properties of interest; (3)identify a possible change to the codon sequence that does not changethe encoded polypeptide; (4) quantify the one or more polynucleotidesequence properties of interest for the polynucleotide sequence afterthe change in codon sequence; (5) accept or reject the change based on afunction of the difference in values for the one or more polynucleotideproperties of interest for the original and the unchanged codonsequences; and (6) repeat this process until a desired value for the oneor more polynucleotide sequence properties of interest is obtained.Searches for codon sequences that have defined values for one or morequantifiable polynucleotide sequence properties can also be performed byevolving the sequence using genetic algorithms or genetic algorithms incombination with monte carlo algorithms, or other stochastic searchessuch as simulated annealing, Boltzmann learning, etc. See for exampleDuda et al., 2001, Pattern Classification, Second Edition, John Wiley &Sons, Inc., New York, which is hereby incorporated by reference hereinfor such purpose.

In preferred embodiments of the invention, the design of polynucleotidesequences with one or more quantifiable polynucleotide sequence propertyis performed using a computer program.

5.4 DESIGN OF CODON VARIANT SETS

The methods disclosed herein may be used to determine at least oneproperty that affects an expression property value of polynucleotides inan expression system. There are many factors that may affect the levelsof expression of a polypeptide by translation of a polynucleotide, orthe levels of expression of soluble polypeptide by translation of apolynucleotide, or the levels of expression of active polypeptide bytranslation of a polynucleotide. Some of these factors can besignificantly influenced by the choice of codons used to encode thepolypeptide, so that the final level of polypeptide expressed, orsoluble polypeptide expressed, or active polypeptide expressed can beincreased 2-fold, or 5-fold or 10-fold or 100-fold by modifying thecodons used to encode a polypeptide. It may not be possible to deducewhich codons will result in improved performance of a polynucleotide byconsidering the biochemistry of the translation process, or of othercellular or biochemical processes that affect translation. Furthermore,different expression systems may differ in the precise details oftranslation (the concentrations of different tRNAs present in a cell, orthe rate at which each tRNA is charged with the appropriate amino acid,for example), so that the codons that may result in high levels ofpolypeptide expression in one expression system may not do so when usedin another expression system.

In cases where it is not possible to assign values to different codonsin an expression system of interest based on a mechanistic understandingof the process, it is possible to elucidate the effect of codon choicesby constructing a plurality of polynucleotides, where the plurality ofpolynucleotides comprises, for example, five or more polynucleotides,ten or more polynucleotides, twenty or more polynucleotides, fifty ormore polynucleotides, 100 or more polynucleotides, between ten and 250polynucleotides, or more than 1000 polynucleotides. In some embodiments,each polynucleotide in the plurality of polynucleotides encodes apolypeptide sequence that is at least seventy-five percent identical, atleast eighty percent identical, at least eighty-five percent identical,at least ninety percent identical, or at least ninety-five percentidentical to at least one other polypeptide sequence encoded by adifferent polynucleotide in the plurality of polynucleotides.

In some embodiments, for each respective amino acid in a plurality ofamino acids comprising five or more amino acids, a relative frequency ofeach of a plurality of synonymous codons for the respective amino acidis varied in a region of each of two or more of the polynucleotides inthe plurality of polynucleotides that encodes a polypeptide sequencethat is at least ninety-five percent identical to the polypeptidesequence encoded by a first polynucleotide. In some embodiments, foreach respective amino acid in a plurality of amino acids comprising fiveor more amino acids, a relative frequency of each of a plurality ofsynonymous codons for the respective amino acid is varied in a region ofeach of two or more of the polynucleotides in the plurality ofpolynucleotides that encodes a polypeptide sequence that is at leastninety-five percent identical to at least one other polypeptide sequenceencoded by a different polynucleotide in the plurality ofpolynucleotides.

In some embodiments, for each respective amino acid in a plurality ofamino acids comprising two or more amino acids, a relative frequency ofeach of a plurality of synonymous codons for the respective amino acidis varied in a region of each of five or more of the polynucleotides inthe plurality of polynucleotides that encodes a polypeptide sequencethat is at least ninety-five percent identical to the polypeptidesequence encoded by a first polynucleotide. In some embodiments, foreach respective amino acid in a plurality of amino acids comprising twoor more amino acids, a relative frequency of each of a plurality ofsynonymous codons for the respective amino acid is varied in a region ofeach of five or more of the polynucleotides in the plurality ofpolynucleotides that encodes a polypeptide sequence that is at leastninety-five percent identical to at least one other polypeptide sequenceencoded by a different polynucleotide in the plurality ofpolynucleotides.

In some embodiments, for each respective amino acid in a plurality ofamino acids comprising between two and fifteen amino acids, a relativefrequency of each of a plurality of synonymous codons for the respectiveamino acid is varied in a region of each of five or more of thepolynucleotides in the plurality of polynucleotides that encodes apolypeptide sequence that is at least eighty, at least eighty-five, atleast ninety, at least ninety-five, at least ninety-eight, or at leastninety-nine percent identical to the polypeptide sequence encoded by afirst polynucleotide. In some embodiments, for each respective aminoacid in a plurality of amino acids comprising between two and fifteenamino acids, a relative frequency of each of a plurality of synonymouscodons for the respective amino acid is varied in a region of each offive or more of the polynucleotides in the plurality of polynucleotidesthat encodes a polypeptide sequence that is at least eighty, at leasteighty-five, at least ninety, at least ninety-five, at leastninety-eight, or at least ninety-nine percent identical to at least oneother polypeptide sequence encoded by a different polynucleotide in theplurality of polynucleotides.

In some embodiments, for each respective amino acid in a plurality ofamino acids comprising five or more amino acids, a relative frequency ofeach of a plurality of synonymous codons for a respective amino acid isvaried in a region of each of five or more of the polynucleotides in theplurality of polynucleotides that encodes a polypeptide sequence that isat least ninety-five percent identical to the polypeptide sequenceencoded by a first polynucleotide. In some embodiments, for eachrespective amino acid in a plurality of amino acids comprising five ormore amino acids, a relative frequency of each of a plurality ofsynonymous codons for a respective amino acid is varied in a region ofeach of five or more of the polynucleotides in the plurality ofpolynucleotides that encodes a polypeptide sequence that is at leastninety-five percent identical to at least one other polypeptide sequenceencoded by a different polynucleotide in the plurality ofpolynucleotides.

As used, herein, computation of percent identity takes full weight ofany insertions in two sequences for which percent identity is computed.To compute percent identity between two sequences, they are aligned andany necessary insertions in either sequence being compared are then madein accordance with sequence alignment algorithms known in the art. Then,the percent identity is computed, where each insertion in eithersequence necessary to make the optimal alignment between the twosequences is counted as a mismatch.

In a preferred embodiment, a first amino acid is encoded a firstplurality of times (e.g., two or more times, three or more times, fouror more times, more than ten times, or between two and one hundredtimes) in both the first polynucleotide and in a second polynucleotidein the plurality of polynucleotides. For example, the first amino acidmay be alanine, or any other naturally occurring amino acid that areencoded by a plurality of synonymous codons including a first codon. Insome embodiments, the plurality of synonymous codons is two or morecodons, three or more codons, four or more codons, five or more codons,or six codons. In some embodiments, this first codon is present in thefirst polynucleotide with a first frequency relative to all other codonsin the plurality of synonymous codons in the first polynucleotide. Forexample, consider the case where there are four synonymous codons forthe first amino acid, where the four synonymous codons include the firstcodon. In some embodiments, the first frequency refers to the percentageof time the first codon is used in a polynucleotide (e.g., the firstpolynucleotide) relative to the percentage of the time all other codonsin the set of four synonymous codons are used in the same polynucleotidein the reading frame that encodes the polypeptide sequence. Thus, if thefirst amino acid appears ten times in the first polynucleotide in thereading frame that encodes the polypeptide sequence under study, and thefirst codon is used to encode three instances of the first amino acid inthe polypeptide sequence, the relative frequency of the first codon inthe first polynucleotide is 0.30.

The first codon is also present in the second polynucleotide with asecond frequency relative to all other codons in the plurality ofsynonymous codons in the second polynucleotide. Further still, the firstfrequency is different than the second frequency. Thus, if the relativefrequency of the first codon in the first polynucleotide is 0.30, thenthe relative frequency of the first codon in the second polynucleotidemust be some value other than 0.30.

In some embodiments, the plurality of nucleotides is constructed usingfrequency lookup tables. Such frequency lookup tables specify theallowed frequency range (e.g., on an absolute basis relative to thetotal number of codons in the coding reading frame or on a relativebasis with respect to the number of synonymous codons in the codingreading frame) for each of a plurality of codons. In fact, in someembodiments, two or more frequency lookup tables can be used toconstruct the plurality of nucleotides. In one exemplary embodiment, thefirst polynucleotide described above is constructed using a firstfrequency lookup table, where the first frequency lookup table specifiesa first target frequency range for the use of the first codon relativeto all other codons in the plurality of synonymous codons in apolynucleotide and where the first frequency described above is withinthe first target frequency range. Further, the second polynucleotidedescribed above is encoded using a second frequency lookup table, wherethe second frequency lookup table specifies a second target frequencyrange for the use of the first codon relative to all other codons in theplurality of synonymous codons in a polynucleotide and where the secondfrequency described above is within the second target frequency range.In this embodiment, the first target frequency range is different fromthe second target frequency target range. For instance, the first targetfrequency range may be 0.25 to 0.75 whereas the second target frequencyrange may be 0.45 to 0.90. Two frequency ranges are considered differentherein if they have at least one different maximum or at least onedifferent minimum. In some embodiments, more than two frequency lookuptables are used to construct the plurality of polynucleotides. Forinstance, referring to the example above where two different codonfrequency tables were used, in some embodiments a third frequency lookuptable specifies a third target frequency range for the use of the firstcodon relative to all other codons in the plurality of synonymous codonsin a third polynucleotide in the plurality of polynucleotides.

In another exemplary aspect of the invention, an embodiment of theconstructing step provides a first frequency lookup table that specifiesa corresponding respective target frequency range for each codon in afirst plurality of codons, each corresponding respective targetfrequency range specifying a target frequency range for a codon in thefirst plurality of codons to be used to encode a corresponding aminoacid in an amino acid sequence relative to all other codons that arecapable of encoding the corresponding amino acid, and where, for eachrespective codon in the first codon frequency table, the constructingstep discussed above further comprises choosing a respective frequencythat the respective codon is to be used to encode the amino acidencodable by the respective codon throughout an amino acid sequenceencoded by the first polynucleotide in the plurality of polynucleotidesrelative to all other codons capable of encoding the amino acid, wherethe respective frequency is within the frequency range specified in thefirst frequency lookup table for the respective codon. A secondfrequency lookup table specifies a corresponding respective targetfrequency range for each codon in a second plurality of codons, eachcorresponding respective target frequency range specifying a targetfrequency range for a codon in the second plurality of codons to be usedto encode a corresponding amino acid in an amino acid sequence relativeto all other codons that are capable of encoding the corresponding aminoacid and where, for each respective codon in the second frequency lookuptable, the constructing step discussed above further comprises choosinga respective frequency that the respective codon is to be used to encodethe amino acid encodable by the respective codon throughout an aminoacid sequence encoded by the second polynucleotide in the plurality ofpolynucleotides relative to all other codons capable of encoding theamino acid, where the respective frequency is within the frequency rangespecified in the second frequency lookup table for the respective codon.In this exemplary embodiment, the first frequency used to encode thefirst codon in the first polynucleotide is within a frequency rangespecified for the first amino acid by the first frequency lookup tableand the second frequency used to encode the first codon in the secondpolynucleotide is within a frequency range specified for the first aminoacid by the second frequency lookup table. In some embodiments the firstplurality of codons of the first frequency lookup table includes codonsthat are not present in the second plurality of codons of the secondfrequency lookup table. In some embodiments, the first plurality ofcodons of the first frequency lookup table is identical to the secondplurality of codons of the second frequency lookup table.

In some embodiments, two or more different frequency lookup tables canbe used to construct a polynucleotide in the plurality ofpolynucleotides. For example, in some embodiments, two differentfrequency lookup tables are used to construct the first polynucleotidedescribed above. In such embodiments, a first set of positions in thefirst polynucleotide are encoded using a first frequency lookup table,where the first frequency lookup table specifies a first targetfrequency range for the use of a predetermined codon, relative to allother codons that are synonymous to the predetermined codon, in thefirst set of positions in the first polynucleotide that encode apredetermined amino acid. Furthermore in these exemplary embodiments, asecond set of positions in the first polynucleotide are encoded using asecond frequency lookup table, where the second frequency lookup tablespecifies a second target frequency range for the use of thepredetermined codon, relative to all other codons that are synonymous tothe predetermined codon, in the second set of positions in the firstpolynucleotide that encode the predetermined amino acid. Further, thefirst set of positions does not include positions in the second set ofpositions and the second set of positions does not include positions inthe first set of positions. An example of a first set of positions areall positions in the reading frame of the polynucleotide that code foran alanine in the N-terminal half of the encoded polypeptide sequence.An example of a second set of positions are all positions in the readingframe of the polynucleotide that code for an alanine in the C-terminalhalf of the encoded polypeptide sequence. In some instances inaccordance with such embodiments, the predetermined amino acid can beencoded by two or more synonymous codons, three or more synonymouscodons, four or more synonymous codons, five or more synonymous codons,or six synonymous codons.

In some embodiments, multiple codon frequency tables are used and eachsuch codon frequency table provides a target frequency range for each ofa plurality of codons (e.g., two or more codons, three or more codons,four or more codons, five or more codons, ten or more codons, fifteen ormore codons, or twenty or more codons). For example, in one example, afirst frequency lookup table specifies a corresponding respective firsttarget frequency range for each codon in a first plurality of codons(e.g., two or more codons, three or more codons, four or more codons,five or more codons, ten or more codons, fifteen or more codons, ortwenty or more codons), each corresponding respective first targetfrequency range specifying a first target frequency range for a codon inthe first plurality of codons to be used to encode a corresponding aminoacid in an amino acid sequence relative to all other codons that arecapable of encoding the corresponding amino acid, and where for eachrespective codon in the first frequency lookup table, the constructingdiscussed above further comprises choosing a respective first frequencythat the respective codon is to be used to encode the amino acidencodable by the respective codon in a predetermined first set ofpositions in the amino acid sequence encoded by the first polynucleotidein the plurality of polynucleotides relative to all other codons capableof encoding the amino acid, where the respective first frequency iswithin the first target frequency range specified in the first frequencylookup table for the respective codon. Further, a second frequencylookup table specifies a corresponding respective second targetfrequency range for each codon in a second plurality of codons, eachcorresponding respective second target frequency range specifying asecond target frequency range for a codon in the second plurality ofcodons to be used to encode a corresponding amino acid in an amino acidsequence relative to all other codons that are capable of encoding thecorresponding amino acid and where, for each respective codon in thesecond frequency lookup table, the constructing discussed above furthercomprises choosing a respective second frequency that the respectivecodon is to be used to encode the amino acid encodable by the respectivecodon in a predetermined second set of positions in the amino acidsequence encoded by the first polynucleotide in the plurality ofpolynucleotides relative to all other codons capable of encoding theamino acid, where the respective second frequency is within the secondtarget frequency range specified in the second frequency lookup tablefor the respective codon. In some embodiments the first plurality ofcodons of the first frequency lookup table includes codons that are notpresent in the second plurality of codons of the second frequency lookuptable. In some embodiments, the first plurality of codons of the firstfrequency lookup table is identical to the second plurality of codons ofthe second frequency lookup table.

In some embodiments, multiple codon frequency tables are used and eachsuch codon frequency table provides a target frequency range for each ofa plurality of codons (e.g., two or more codons, three or more codons,four or more codons, five or more codons, ten or more codons, fifteen ormore codons, or twenty or more codons), where the frequency range for agiven codon is relative to all other expressed codons in the readingframe of a polynucleotide, not just synonymous codons. For example, oneembodiment provides a first frequency lookup table that specifies acorresponding respective first target frequency range for each codon ina first plurality of codons, each corresponding respective first targetfrequency range specifying a first target frequency range for a codon inthe first plurality of codons to be used to encode a corresponding aminoacid in an amino acid sequence relative to all other expressed codons inthe polynucleotide, and where for each respective codon in the firstcodon frequency table, the constructing step described above furthercomprises choosing a respective first frequency that the respectivecodon is to be used to encode the amino acid encodable by the respectivecodon throughout the amino acid sequence encoded by the firstpolynucleotide in the plurality of polynucleotides relative to all otherexpressed codons in the first polynucleotide, where the respective firstfrequency is within the first target frequency range specified in thefirst frequency lookup table for the respective codon Furthermore, asecond frequency lookup table specifies a corresponding respectivesecond target frequency range for each codon in a second plurality ofcodons, each corresponding respective second target frequency rangespecifying a second target frequency range for a codon in the secondplurality of codons to be used to encode a corresponding amino acid inan amino acid sequence relative to all other expressed codons in thepolynucleotide, and where for each respective codon in the secondfrequency lookup table, the constructing discussed above furthercomprises choosing a respective second frequency that the respectivecodon is to be used to encode the amino acid encodable by the respectivecodon throughout the amino acid sequence encoded by said secondpolynucleotide in the plurality of polynucleotides relative to all otherexpressed codons in the second polynucleotide, where the secondrespective frequency is within the second target frequency rangespecified in the second frequency lookup table for the respective codon.In this embodiment the first frequency used to encode a first codon inthe first polynucleotide is within a first target frequency rangespecified for the first amino acid by the first frequency lookup table.Furthermore, the second frequency used to encode the first codon presentin the second polynucleotide is within a second target frequency rangespecified for the first amino acid by the second frequency lookup table.

In the inventive methods, each respective polynucleotide in theplurality of polynucleotides is expressed individually in an expressionsystem. In some embodiments, this involves synthesizing each of thepolynucleotides in the plurality of polynucleotides, inserting eachpolynucleotide into a separate expression vector, and expressing thepolynucleotide in the expression vector separately in the expressionsystem. Examples of expression systems that can be used have been givenabove and include, for example, E. coli and baculovirus and yeast andmammalian tissue culture and plants such as maize or soybean.

Next, an expression property value of each respective polynucleotide inthe plurality of polynucleotides in the expression system is measured,thereby determining at least one property that affects an expressionproperty of polynucleotides in the expression system, where the at leastone property is an effect that a frequency of use of one or more codonsin a plurality of naturally occurring codons has on the expressionproperty values of polynucleotides in the expression system.

Standard techniques may be utilized to measure the expression propertyvalue of each respective polynucleotide in the plurality ofpolynucleotides. For example, standard techniques can be employed using,e.g., immunoassays such as, for example Western blot,immunoprecipitation followed by sodium dodecyl sulfate polyacrylamidegel electrophoresis, (SDS-PAGE), immunocytochemistry, and the like todetermine an expression property value of a respective polynucleotide(e.g., an amount of a protein encoded by the respective polynucleotide)in the plurality of polynucleotides present in an expression system.Other methods for detection of specific polypeptides include massspectroscopy and mass spectroscopy of protein samples that have beentreated with one or more site specific proteases to produce polypeptidefragments which can be uniquely identified by mass spectroscopy. Oneexemplary agent for detecting a protein of interest is an antibodycapable of specifically binding to a protein of interest, preferably anantibody detectably labeled, either directly or indirectly.

For such detection methods, if desired, a protein from the expressionsystem to be analyzed can easily be isolated using techniques which arewell known to those of skill in the art. Protein isolation methods can,for example, be such as those described in Harlow and Lane, 1988,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press(Cold Spring Harbor, N.Y.), which is incorporated by reference herein inits entirety.

In certain embodiments, methods of detection of the protein expressedfrom a respective polynucleotide in the plurality of polynucleotides inthe expression system involve detection via interaction with aprotein-specific antibody. For example, antibodies directed to a proteinof interest (e.g., a protein expressed from a respective polynucleotidein the plurality of polynucleotides). Antibodies can be generatedutilizing standard techniques well known to those of skill in the art.In specific embodiments, antibodies can be polyclonal, or morepreferably, monoclonal. An intact antibody, or an antibody fragment(e.g., scFv, Fab or F(ab′)₂) can, for example, be used.

For example, antibodies, or fragments of antibodies, specific for aprotein of interest can be used to quantitatively or qualitativelydetect the presence of a protein. This can be accomplished, for example,by immunofluorescence techniques. Antibodies (or fragments thereof) can,additionally, be employed histologically, as in immunofluorescence orimmunoelectron microscopy, for in situ detection of a protein ofinterest. In situ detection can be accomplished by applying to anexpression system (used to express a protein from a respectivepolynucleotide in the plurality of polynucleotides) a labeled antibodythat is directed to the protein. The antibody (or fragment) ispreferably applied by overlaying the antibody (or fragment) onto theexpression system.

Immunoassays for a protein of interest typically comprise incubating anexpression system with a detectably labeled antibody capable ofidentifying a protein of interest, and detecting the bound antibody byany of a number of techniques well-known in the art. The term “labeled”can refer to direct labeling of the antibody via, e.g., coupling (e.g.,physically linking) a detectable substance to the antibody, and can alsorefer to indirect labeling of the antibody by reactivity with anotherreagent that is directly labeled. Examples of indirect labeling includedetection of a primary antibody using a fluorescently labeled secondaryantibody.

The expression system in which a respective polynucleotide in theplurality of polynucleotides is expressed can be brought in contact withand immobilized onto a solid phase support or carrier such asnitrocellulose, or other solid support which is capable of immobilizingcells, cell particles or soluble proteins. The support can then bewashed with suitable buffers followed by treatment with the detectablylabeled fingerprint gene-specific antibody. The solid phase support canthen be washed with the buffer a second time to remove unbound antibody.The amount of bound label on solid support can then be detected byconventional methods.

By “solid phase support or carrier” is intended any support capable ofbinding an antigen or an antibody. Well-known supports or carriersinclude glass, polystyrene, polypropylene, polyethylene, dextran, nylon,amylases, natural and modified celluloses, polyacrylamides andmagnetite. The nature of the carrier can be either soluble to someextent or insoluble for the purposes of the present invention. Thesupport material can have virtually any possible structuralconfiguration so long as the coupled molecule is capable of binding toan antigen or antibody. Thus, the support configuration can bespherical, as in a bead, or cylindrical, as in the inside surface of atest tube, or the external surface of a rod. Alternatively, the surfacecan be flat such as a sheet, test strip, etc. Preferred supports includepolystyrene beads. Those skilled in the art will know many othersuitable carriers for binding antibody or antigen, or will be able toascertain the same by use of routine experimentation.

One of the ways in which an antibody specific for a protein of interestcan be detectably labeled is by linking the same to an enzyme and use inan enzyme immunoassay (EIA) (Voller, 1978, “The Enzyme LinkedImmunosorbent Assay (ELISA)”, Diagnostic Horizons 2:1-7, MicrobiologicalAssociates Quarterly Publication, Walkersville, Md.; Voller et al.,1978, J. Clin. Pathol. 31:507-520; Butler, J. E., 1981, Meth. Enzymol.73:482-523; Maggio (ed.), 1980, Enzyme Immunoassay, CRC Press, BocaRaton, Fla.; Ishikawa et al., (eds.), 1981, Enzyme Immunoassay, KgakuShoin, Tokyo, each of which is hereby incorporated by reference in itsentirety). The enzyme which is bound to the antibody will react with anappropriate substrate, preferably a chromogenic substrate, in such amanner as to produce a chemical moiety which can be detected, forexample, by spectrophotometric, fluorimetric or by visual means. Enzymeswhich can be used to detectably label the antibody include, but are notlimited to, malate dehydrogenase, staphylococcal nuclease,delta-5-steroid isomerase, yeast alcohol dehydrogenase,alpha-glycerophosphate, dehydrogenase, triose phosphate isomerase,horseradish peroxidase, alkaline phosphatase, asparaginase, glucoseoxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase. The detection can be accomplished by colorimetricmethods which employ a chromogenic substrate for the enzyme. Detectioncan also be accomplished by visual comparison of the extent of enzymaticreaction of a substrate in comparison with similarly prepared standards.

Detection can also be accomplished using any of a variety of otherimmunoassays. For example, by radioactively labeling the antibodies orantibody fragments, it is possible to detect a protein of interestthrough the use of a radioimmunoassay (RIA) (see, for example,Weintraub, 1986, Principles of Radioimmunoassays, Seventh TrainingCourse on Radioligand Assay Techniques, The Endocrine Society, which ishereby incorporated by reference herein). The radioactive isotope (e.g.,¹²⁵I, ¹³¹I, ³⁵S or ³H) can be detected by such means as the use of agamma counter or a scintillation counter or by autoradiography.

It is also possible to label the antibody with a fluorescent compound.When the fluorescently labeled antibody is exposed to light of theproper wavelength, its presence can then be detected due tofluorescence. Among the most commonly used fluorescent labelingcompounds are fluorescein isothiocyanate, rhodamine, phycoerythrin,phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.

The antibody can also be detectably labeled using fluorescence emittingmetals such as ¹⁵²Eu, or others of the lanthanide series. These metalscan be attached to the antibody using such metal chelating groups asdiethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraaceticacid (EDTA).

The antibody also can be detectably labeled by coupling it to achemiluminescent compound. The presence of the chemiluminescent-taggedantibody is then determined by detecting the presence of luminescencethat arises during the course of a chemical reaction. Examples ofparticularly useful chemiluminescent labeling compounds are luminol,isoluminol, theromatic acridinium ester, imidazole, acridinium salt andoxalate ester. Likewise, a bioluminescent compound can be used to labelthe antibody. Bioluminescence is a type of chemiluminescence found inbiological systems in, which a catalytic protein increases theefficiency of the chemiluminescent reaction. The presence of abioluminescent protein is determined by detecting the presence ofluminescence. Important bioluminescent compounds for purposes oflabeling are luciferin, luciferase and aequorin.

In some embodiments, a protein chip assay (e.g., THE PROTEINCHIP®Biomarker System, Ciphergen, Fremont, Calif.) is used to measure anexpression property value of a respective polynucleotide in theexpression system. See also, for example, Lin, 2004, Modern Pathology,1-9; Li, 2004, Journal of Urology 171, 1782-1787; Wadsworth, 2004,Clinical Cancer Research, 10, 1625-1632; Prieto, 2003, Journal of LiquidChromatography & Related Technologies 26, 2315-2328; Coombes, 2003,Clinical Chemistry 49, 1615-1623; Mian, 2003, Proteomics 3, 1725-1737;Lehre et al., 2003, BJU International 92, 223-225; and Diamond, 2003,Journal of the American Society for Mass Spectrometry 14, 760-765, eachof which is hereby incorporated by reference in its entirety. Proteinchip assays (protein microarrays) are commercially available. Forexample, Ciphergen (Fremont, Calif.) markets the PROTEINCHIP® SystemSeries 4000 for quantifying proteins in a sample. Furthermore,Sigma-Aldrich (Saint Lewis, Mo.) sells a number of protein microarraysincluding the PANORAMA™ Human Cancer v1 Protein Array, the PANORAMA™Human Kinase v1 Protein Array, the PANORAMA™ Signal TransductionFunctional Protein Array, the PANORAMA™ AB Microarray—Cell SignalingKit, the PANORAMA™ AB Microarray—MAPK and PKC Pathways kit, thePANORAMA™ AB Microarray—Gene Regulation I Kit, and the PANORAMA™ ABMicroarray—p53 pathways kit. Further, TeleChem International, Inc.(Sunnyvale, Calif.) markets a Colorimetric Protein Microarray Platformthat can perform a variety of micro multiplexed protein microarrayassays including microarray based multiplex ELISA assays. See also,MacBeath and Schreiber, 2000, “Printing Proteins as Microarrays forHigh-Throughput Function Determination,” Science 289, 1760-1763, whichis hereby incorporated by reference herein in its entirety.

In some embodiments, a bead assay is used to measure an expressionproperty value of a respective polynucleotide in the expression system.One such bead assay is the Becton Dickinson Cytometric Bead Array (CBA).CBA employs a series of particles with discrete fluorescence intensitiesto simultaneously detect multiple soluble analytes. CBA is combined withflow cytometry to create a multiplexed assay. The Becton Dickinson CBAsystem, as embodied for example in the Becton Dickinson HumanInflammation Kit, uses the sensitivity of amplified fluorescencedetection by flow cytometry to measure soluble analytes in aparticle-based immunoassay. Each bead in a CBA provides a capturesurface for a specific protein and is analogous to an individuallycoated well in an ELISA plate. The BD CBA capture bead mixture is insuspension to allow for the detection of multiple analytes in a smallvolume sample.

In some embodiments the multiplex analysis method described in U.S. Pat.No. 5,981,180, herein incorporated by reference in its entirety, and inparticular for its teachings of the general methodology, beadtechnology, system hardware and antibody detection, is used to measurean expression property value of a respective polynucleotide in theexpression system. For this analysis, a matrix of microparticles issynthesized, where the matrix consists of different sets ofmicroparticles. Each set of microparticles can have thousands ofmolecules of a distinct antibody capture reagent immobilized on themicroparticle surface and can be color-coded by incorporation of varyingamounts of two fluorescent dyes. The ratio of the two fluorescent dyesprovides a distinct emission spectrum for each set of microparticles,allowing the identification of a microparticle a set following thepooling of the various sets of microparticles. U.S. Pat. Nos. 6,268,222and 6,599,331 also are incorporated herein by reference in theirentirety, and in particular for their teachings of various methods oflabeling microparticles for multiplex analysis.

In some embodiments an assay that distinguishes between soluble andinsoluble polypeptides is used to measure an expression property valueof a respective polynucleotide in the expression system. Such assays mayincorporate centrifugation or filtration or chromatographic steps toseparate soluble polypeptides encoded by a polynucleotide from insolublepolypeptides encoded by a polynucleotide.

In some embodiments, the frequency of use of one or more codons is therelative frequency of use of the one or more codons with respect to theuse of synonymous codons in the reading frame of each of thepolynucleotides in the plurality of polynucleotides that encode thepolypeptide sequence under study. For example, consider the case wherethe one or more codons is in fact, a single codon. In this case, thefrequency of use of the one codon refers to the relative frequency withwhich the one codon is used to encode all instances of the amino acidcorresponding to the one codon in the reading frame of each ofpolynucleotides that encodes the polypeptide under study, relative tothe use of all other synonymous codons in the reading frame of each ofpolynucleotides that encodes the polypeptide under study.

In some embodiments, the frequency of use of one or more codons is theabsolute frequency of use of the one or more codons with respect to thetotal number of codons in the reading frame of each of thepolynucleotides in the plurality of polynucleotides that encode thepolypeptide sequence under study. For example, consider the case wherethe one or more codons is, in fact, a single codon. In this case, theabsolute frequency of use of the one codon refers to the frequency withwhich the one codon is used in the reading frame of each ofpolynucleotides that encodes the polypeptide under study, relative tothe total number of all codons in the reading frame of each ofpolynucleotides that encodes the polypeptide under study.

In some embodiments the expression property value of the respectivepolynucleotide in the plurality of polynucleotides in the expressionsystem is a total amount of protein encoded by the respectivepolynucleotide that is expressed in the expression system in apredetermined period of time. In some embodiments, the expressionproperty value of the respective polynucleotide in the expression systemis a total amount of active protein encoded by the respectivepolynucleotide that is expressed in the expression system in apredetermined period of time. In some embodiments, the expressionproperty of the respective polynucleotide in the expression system is atotal amount of soluble protein encoded by the respective polynucleotidethat is expressed in the expression system in a predetermined period oftime. As used here, the predetermined period of time is applicationdependent and generally refers to a period of time after thepolynucleotide has been introduced into the expression system (e.g., bytransfection of an expression vector). For example, as used here, thepredetermined period of time can be five minutes or more, twenty minutesor more, one hour or more, four hours or more, or less than two daysafter the respective polynucleotide has been introduced into theexpression system and the expression system has been put underconditions that permit protein expression.

Another aspect provides a method of determining at least one propertythat affects an expression property value of polynucleotides in anexpression system, the method comprising constructing a plurality ofpolynucleotides, wherein the plurality of polynucleotides comprises fiveor more polynucleotides, ten or more polynucleotides, twenty or morepolynucleotides, between five and one hundred polynucleotides, or morethan one thousand polynucleotides, each polynucleotide in the pluralityof polynucleotides encoding a polypeptide sequence that is at leastninety-five percent identical to at least one other polypeptide sequenceencoded by a different polynucleotide in the plurality ofpolynucleotides where (i) a first amino acid is encoded a firstplurality of times in both the first polynucleotide and in a secondpolynucleotide in the plurality of polynucleotides, (ii) the first aminoacid is encodable by a plurality of synonymous codons including a firstcodon, (iii) the first codon is present in the first polynucleotide witha first frequency, (iv) the first codon is present in the secondpolynucleotide with a second frequency, and (v) the first frequency isdifferent than the second frequency. Further in the method, eachrespective polynucleotide in the plurality of polynucleotides isexpressed individually in the expression system. Further in the method,an expression property value of each respective polynucleotide in theplurality of polynucleotides in the expression system is measuredthereby determining at least one property that affects an expressionproperty value of polynucleotides in the expression system, where the atleast one property is an effect that a frequency of use of one or morecodons in a plurality of naturally occurring codons has on theexpression property values of polynucleotides in the expression system,where a first frequency lookup table specifies a correspondingrespective first target frequency range for each codon in a firstplurality of codons, each corresponding respective first targetfrequency range specifying a first target frequency range for a codon inthe first plurality of codons to be used to encode a corresponding aminoacid in an amino acid sequence relative to all other expressed codons inthe polynucleotide, and wherein for each respective codon in the firstfrequency lookup table, the constructing further comprises choosing arespective first frequency that the respective codon is to be used toencode the amino acid encodable by the respective codon throughout theamino acid sequence encoded by said first polynucleotide in theplurality of polynucleotides relative to all other expressed codons inthe first polynucleotide, wherein the respective first frequency iswithin the first target frequency range specified in the first frequencylookup table for the respective codon; and a second frequency lookuptable specifies a corresponding respective second target frequency rangefor each codon in a second plurality of codons, each correspondingrespective second target frequency range specifying a second targetfrequency range for a codon in the second plurality of codons to be usedto encode a corresponding amino acid in an amino acid sequence relativeto all other expressed codons in the polynucleotide, and wherein foreach respective codon in the second frequency lookup table, theconstructing further comprises choosing a respective second frequencythat the respective codon is to be used to encode the amino acidencodable by the respective codon throughout the amino acid sequenceencoded by said second polynucleotide in the plurality ofpolynucleotides relative to all other expressed codons in the secondpolynucleotide, where the second respective frequency is within thesecond target frequency range specified in the second frequency lookuptable for the respective codon. In some embodiments, for each respectiveamino acid in a plurality of amino acids comprising five or more aminoacids, a relative frequency of each of a plurality of synonymous codonsfor a respective amino acid is varied in a region of each of five ormore of the polynucleotides in the plurality of polynucleotides thatencodes a polypeptide sequence that is at least ninety-five percentidentical to at least one other polypeptide sequence encoded by adifferent polynucleotide in the plurality of polynucleotides

The plurality of polynucleotides, in which each polynucleotide encodesthe same polypeptide sequence (or variants thereof having the requisitepercent identity and described above) but differ in the codons used toencode that polypeptide sequence at one or more positions within thepolynucleotide is referred to herein as a codon variant set. Thesequences of the polynucleotides comprising the codon variant set, orone or more of the polynucleotide sequence properties of thepolynucleotide sequences in the codon variant set (properties includingbut not limited to the codon bias, the relative or absolute frequencywith which specific codons are used, the GC content of thepolynucleotide, the predicted mRNA secondary structure of thepolynucleotide) can then be correlated with one or more properties(e.g., expression properties) of the polynucleotide such as the levelsof expression of a polypeptide by translation of the polynucleotide inan expression system, or the levels of expression of soluble polypeptideby translation of the polynucleotide in an expression system, or thelevels of expression of a biologically or chemically active polypeptideby translation of the polynucleotide in an expression system. Suchcorrelation can be achieved manually. For example, an expressionproperty of each of the polynucleotide in the plurality ofpolynucleotides can be compared to the codon usage in each of thepolynucleotides in order to ascertain a relationship between codon usage(e.g., codon frequency) and the expression property. Such correlationcan also be achieved using pattern classification methods or statisticalmethods. Examples of pattern classification methods or statisticalmethods include, but are not limited to linear regression, non-linearregression, logistic regression, multivariate data analysis,classification using a regression tree, partial least squares projectionto latent variables, computation of a neural network, computation of aBayesian model, computation of a generalized additive model, use of asupport vector machine, or modeling comprising boosting or adaptiveboosting. See, for example, Duda et al, 2001, Pattern Classification,Second Edition, John Wiley & Sons, Inc., New York; Hastie, 2003, TheElements of Statistical Learning, Springer, N.Y.; and Agresti 1996, AnIntroduction to Categorical Data Analysis, John Wiley & Sons, New York,each of which is hereby incorporated by reference herein for suchpurpose. Such modeling or correlation can then be used to assign valuesfor the different codons in the expression system of interest. Thedesign and synthesis of a codon variant set and measurement of anexpression property of the polynucleotides within a codon variant setfor the purpose of evaluating different codon choices within anexpression system is an aspect of the present invention.

The method of systematic variation of codon usage and analysis ofexpression is conceptually quite different from previous methodsdescribed in the art, and is an aspect of the present invention. Theseprevious methods have used weak correlations between the codon biasesfound in and the expression levels observed for natural genes to deriverules for heterologous gene design. Such data from natural genes arevery unlikely to provide a basis from which to accurately model theeffects of codon choices within the open reading frame upon expression.This is because there is no systematic variation of codon use in naturalgenes, and because natural genes also include many other differences insequence elements that have important effects upon expression, such asdifferences in promoter sequences for initiation of transcription,differences in sequences at and near the initiation codon includingribosome binding sites, internal ribosome entry sites and Kozakconsensus sequences, differences in RNA-stabilizing or destabilizingmotifs outside the open reading frame. In contrast the method of thepresent invention can be performed without assumptions regarding thenatural codon preferences of the expression host, or the underlyingmechanism of such preference. Instead, the expression system isinterrogated with systematically varied sets of sequences andmeasurements of the expression properties of interest to determine thecodon biases that result in desired expression properties. This methodmay be applied to any expression system as well as to identify anoptimal bias for high expression in multiple systems if a polynucleotideis to be expressed in different systems.

5.4.1 Constructing Codon Frequency Lookup Tables for Design ofPolynucleotides in Codon Variant Sets

A plurality of polynucleotide sequences for sequence-expression modelingmay be obtained by varying one or more design parameters during thedesign of each polynucleotide within the plurality of polynucleotidesequences.

In one preferred embodiment, polynucleotide sequences forsequence-expression modeling may be designed using a set of differentvariant design codon frequency lookup tables. One variant design codonfrequency lookup table can be used for designing each polynucleotidesequence in the codon variant set (plurality of polynucleotidesequences), for example by probabilistically selecting codons accordingto the frequency in the variant design codon frequency lookup table, orby using genetic algorithms or monte carlo algorithms to produce apolynucleotide sequence that conforms to the variant design codonfrequency lookup table.

In a preferred embodiment of the invention, selection of the relativesynonymous codon frequencies for an amino acid in a variant design codonfrequency lookup table can be performed as a function of the codonbiases for that amino acid within an initial codon bias matrix. Aninitial codon bias matrix is a table comprising one or more codon biasesfor each amino acid. There may be more different codon biases for oneamino acid than another. Values for codon bias in an initial codon biasmatrix may be drawn from codon bias tables derived from natural genesequences, or calculated in other ways, for example they may becalculated based upon known biochemical processes such as the rate ofrecharging (amino acylation) of tRNAs, or they may be calculated basedupon the GC content of the codon, or they may be calculated based uponexperimentally determined codon preferences for an expression system ofinterest.

For example, consider using the codon bias values from the genomicsequences of Saccharomyces cerevisiae open reading frames and those fromhighly expressed E. coli genes for the initial codon bias matrix. Thecodon bias for Tyr in the E. coli codon bias table is TAC 0.65 and TAT0.35. The codon bias for Tyr in the Saccharomyces cerevisiae codon biastable is TAC 0.44 and TAT 0.56. The function by which a codon bias forTyr may be calculated for a variant design codon frequency lookup tablehas many possible forms. In one form, the new bias for Tyr could simplybe selected from one of the original biases, in this case either the E.coli bias or the S cerevisiae bias. In another form, the new bias forTyr could be selected probabilistically based on a distribution betweenthe two biases in the initial codon bias matrix. In another form, thenew bias for Tyr could use exclusively the codon that is used most onaverage (in this case TAC) or exclusively the codon which is used leaston average (in this case TAT). In another form, one or more additionalcodon biases for Tyr may be calculated as intermediates between thecodon biases from the initial codon bias matrix and the new bias for Tyrselected from one of these. In another form, an additional codon biasfor Tyr may be calculated as each codon represented equally; this “flat”codon bias may be used in combination with any of the other forms of thecodon bias calculation function. One skilled in the art will readilyappreciate that there are many ways to calculate a new codon bias usingtwo or more initial codon biases. Any of these methods may be used tocalculate the codon bias for each amino acid to be used in constructinga variant design codon frequency lookup table. Further, the codon biasin a variant design codon frequency lookup table can be selected in thesame way for each amino acid, or it may be selected differently fordifferent amino acids.

The aim when designing a plurality of polynucleotides to interrogate acodon space is to obtain codon usages that are distributed in such a waythat a large amount of information can subsequently be extracted fromsequence-expression relationships. In this respect, the design of codonvariant sets has common elements with the design of experimentaldatasets from a diverse range of other disciplines including agricultureand engineering. Methods to optimize experimental datasets (experimentaldesign or design of experiment: DOE) are described by Sir R. A. Fisherin 1920 (Fisher, The Design of Experiments, MacMillan PublishingCompany; 9th edition, 1971). Plackett and Burman developed the ideafurther with the introduction of screening designs (e.g., Plackett etal., 1946, Biometrika 33: 305-325), and Taguchi subsequently introducedthe orthogonal matrix (Taguchi, 1986, Introduction to QualityEngineering, Asian Productivity Organization, Distributed by AmericanSupplier Institute Inc., Dearborn, Mich.).

In a preferred embodiment, one or more values for initial codon biasesfor each amino acid are selected into an initial codon bias matrix. Oneamino acid may have more discrete values for initial codon biases thananother amino acid. Variant design codon frequency lookup tables arethen constructed using experimental design techniques to select thecodon bias for each amino acid in a way that maximizes the informationcontent of the set of variant design codon frequency lookup tables.These experimental design methods include, but are not limited to,complete factorial design, 2^(k) factorial design, 2^(k) fractionalfactorial design, central composite, latin squares, greco-latin squares,Plackett-Burmann designs, Taguchi design, and combinations thereof. See,for example, Box et al, 1978, Statistics for Experimenters. New York,Wiley, for examples of such techniques that can be used to construct adesigned codon variant set from the initial set of variant design codonfrequency lookup tables selected to test a maximum number of codonbiases in a minimal number of codon variants.

A variation of the above method is to require (i) that for each aminoacid, each possible codon bias for that amino acid in the initial codonbias matrix be used an approximately equal number of times in thevariant design codon frequency lookup table set, and (ii) that as manydifferent combinations of codon biases for different amino acids (e.g.codon bias pairs) as possible be tested. The solution to such a problemof finding variants with the constraints mentioned here is known as acoverage problem. The coverage problem is NP-hard. Therefore greedy andother forms of approximate solutions are used to solve the NP-hardproblems in the present invention. For instance, in some embodiments,the algorithms described in Gandhi et al., 2001, Lecture Notes inComputer Science 2076: 225 are used.

As in example, in some embodiments, the desired set of sequences can beevolved using monte carlo algorithms and/or genetic algorithms tomaximize the number of pairs in the plurality of nucleotides. Geneticalgorithms are described in Section 7.5.1 of Duda et al, 2001, PatternClassification, Second Edition, John Wiley & Sons, Inc., New York, whichis hereby incorporated by reference. Further, similar algorithms can beused to expand the coverage problem to maximize the number of triplets,quadruplets and so on.

An exemplary code for maximizing the codon bias pairs using anevolutionary coverage algorithm is shown below:

Let n be the number of variant design codon frequency lookup tables tobe constructed.

First, create n variant design codon frequency lookup tables, for eachamino acid codon bias in each variant design codon frequency lookuptable randomly select an initial codon bias for that amino acid drawnfrom the initial codon bias matrix. To obtain a set of variant designcodon frequency lookup tables where the codon bias for each amino acidis approximately equally distributed between the possibilities containedin the codon bias matrix, and where the number of codon bias pairs aremaximized; first

for 10000 iterations

{

-   -   i. Calculate the frequency with which the codon bias for each        individual amino acid appearing in the variant design codon        frequency lookup tables are drawn from each possible value in        the initial codon bias matrix;    -   ii. Randomly choose one variant design codon frequency lookup        table;    -   iii. Randomly choose one individual amino acid;    -   iv. Randomly change the codon bias in the selected variant        design codon frequency lookup tables for the selected individual        amino acid to a different codon bias for this amino acid drawn        from the initial codon bias matrix;    -   v. Calculate the frequency with which the codon bias for each        individual amino acid appearing in the new set of variant design        codon frequency lookup tables are drawn from each initial codon        frequency lookup table;    -   vi. If the frequencies from v) are more even than the        frequencies from i), accept the changes from step iv), else,        dismiss the changes and retain original values.

}

Output an evolved set of variant design codon frequency lookup tables,then

for 10000 iterations

{

-   -   vii. Count the number of distinct codon bias pairs seen within        the set of variant design codon frequency lookup tables;    -   viii. Randomly choose two variant design codon frequency lookup        tables;    -   ix. Randomly choose one individual amino acid;    -   x. Randomly swap the codon biases for the one individual amino        acid between the two selected variant design codon frequency        lookup tables;    -   xi. Count the number of distinct codon bias pairs seen within        the new set of variant design codon frequency lookup tables;    -   xii. If the count from xi) is greater than count from vii),        accept the changes to the variants from step x), else, dismiss        the changes and retain original values.

}

In other embodiments the required distributions may be different, forexample the constraint could be that for each amino acid, each possiblecodon bias for that amino acid in the initial codon bias matrix be usedin a distribution other than an approximately equal number of times inthe variant design codon frequency lookup table set. In another example,the constraint could be that that as many different combinations ofcodon biases for only a subset of the different amino acids (e.g. codonbias pairs) as possible be tested. One skilled in the art willappreciate that evolutionary algorithms can be used to design a set ofvariant design codon frequency lookup tables when these constraints arevaried. It is also possible to create codon variant sets where the codonbias for one amino acid is optimized by systematic variation of thecodons for that amino acid without varying codon biases for the otheramino acids.

5.4.2 Other Methods for Designing Polynucleotides for Codon Variant Sets

Codon variant sets may be constructed to explore other parameters withinthe open reading frame that may affect expression properties of apolynucleotide. In a preferred embodiment this can be done by a simplemodification of the method described for constructing variant designcodon frequency lookup tables. Instead of codon frequency lookup tables,tables can be constructed that describe target values for anyquantifiable polynucleotide sequence properties, including but notlimited to the frequency of any sequence element, (a sequence elementcan optionally be defined in terms of both a sequence and a readingframe relative to the reading frame of the encoded polypeptide), whichincludes codons (as described above). Particularly useful sequenceelements include a nucleotide triplet in the +3 reading frame, whichencodes the “wobble” or third base of one codon and the first two basesof the following codon, and a nucleotide hexamer in the +1 readingframe, which encodes an adjacent pair of codons (a “codon pair”). Otherquantifiable sequence properties include GC content, mRNA secondarystructures in particular regions of an mRNA produced by thepolynucleotide for example covering the start codon, the degree ofsequence identity to a reference sequence, the presence of ribosomebinding sites, polyadenylation signals, polynucleotide splice signals,the annealing temperature for a sub-sequence of predetermined lengthwithin the polynucleotide for any other sub-sequence within thepolynucleotide, repeated sequence elements or homopolymer stretches.Variation in GC %, either of the entire codon or simply at one position(e.g., the more variable 3′ terminal nucleotide) is a useful way to varyoverall bias and can add diversity in design. These variant designpolynucleotide sequence property tables can be used in a similar way tovariant design codon frequency lookup tables: a polynucleotide can bedesigned such that it conforms to the values for quantifiable sequenceproperties described in one of the polynucleotide sequence propertytables.

This may be achieved for example using an evolutionary algorithm thatperforms the following: (1) select an initial polynucleotide sequence toencode a polypeptide; (2) quantify the one or more polynucleotidesequence properties of interest; (3) identify a possible change to thepolynucleotide sequence that does not change the encoded polypeptide;(4) quantify the one or more polynucleotide sequence properties ofinterest for the polynucleotide sequence after the change inpolynucleotide sequence; (5) accept or reject the change based on afunction of the difference in values for the one or more polynucleotideproperties of interest for the original and the unchanged codonsequences; and (6) repeat this process until a desired value for the oneor more polynucleotide sequence properties of interest is obtained. Itis also possible to combine designs for polynucleotides in which aspecific codon frequency can be combined with one or more other desiredpolynucleotide sequence properties. One example of how this may be doneis:

First, select a polynucleotide sequence encoding the target polypeptide,

then for 10000 iterations

{

-   -   i. calculate the codon bias for each amino acid in the        polynucleotide;    -   ii. compare the codon bias for each amino acid in the        polynucleotide with the codon bias in the variant design codon        frequency lookup table;    -   iii. randomly choose one codon in the polynucleotide;    -   iv. randomly change the selected codon for a different codon        encoding the same amino acid;    -   v. calculate the codon bias for each amino acid in the modified        polynucleotide;    -   vi. compare the codon bias for each amino acid in the        polynucleotide with the codon bias in the variant design codon        frequency lookup table;    -   vii. if the match between the variant design codon frequency        lookup table and the codon bias in the modified polynucleotide        calculated in vii) is better than the match between the variant        design codon frequency lookup table and the codon bias in the        original polynucleotide calculated in iii), accept the changes        from step v), else, dismiss the changes and retain original        values.

}

Output an evolved sequence, then

for 10000 iterations

{

-   -   viii. calculate a value for each of the quantifiable        polynucleotide properties of interest for the polynucleotide;    -   ix. compare the values calculated in viii) with the values for        the properties described in the polynucleotide sequence property        table;    -   x. randomly choose two codons for the same amino acid within the        polynucleotide;    -   xi. exchange the two codons;    -   xii. calculate a value for each of the quantifiable        polynucleotide properties of interest for the modified        polynucleotide;    -   xiii. compare the values calculated in xii) with the values for        the properties described in the polynucleotide sequence property        table;    -   xiv. if the match between the values in the polynucleotide        sequence property table and those calculated for the modified        polynucleotide calculated in xii) is better than the match        between the polynucleotide sequence property table and those        calculated for the original polynucleotide calculated in ix),        accept the changes from step x), else, dismiss the changes and        retain original values.

}

Codons may be replaced more generally with any sequence element, forexample by using an evolutionary algorithm that performs the following:(1) select an initial polynucleotide sequence to encode a polypeptide;(2) quantify the one or more polynucleotide sequence properties ofinterest; (3) identify a possible change to the polynucleotide sequencethat does not change the encoded polypeptide; (4) quantify the one ormore polynucleotide sequence properties of interest for thepolynucleotide sequence after the change in polynucleotide sequence; (5)accept or reject the change based on a function of the difference invalues for the one or more polynucleotide properties of interest for theoriginal and the unchanged codon sequences; and (6) repeat this processuntil a desired value for the one or more polynucleotide sequenceproperties of interest is obtained. It is also possible to combinedesigns for polynucleotides in which a specific codon frequency can becombined with one or more other desired polynucleotide sequenceproperties. One example of how this may be done is:

First, select a polynucleotide sequence encoding the target polypeptide,

then for 10000 iterations

{

-   -   i. obtain a sequence element frequency lookup table        corresponding to an expression system, where the frequency        lookup table comprises a plurality of sequence elements and a        plurality of frequency ranges, each frequency range in the        plurality of frequency ranges is for a corresponding sequence        element in the plurality of sequence elements, each respective        frequency range in the plurality of frequency ranges specifies a        range of frequencies that the sequence element corresponding to        the respective frequency range can occur in a polynucleotide        that is to be expressed in the expression system, and each        sequence element in the plurality of sequence elements is        optionally associated with a frame designation in the frequency        lookup table    -   ii. calculate the frequency with which each sequence element is        present in its designated frame (if any) in the polynucleotide;    -   iii. compare the frequencies for each sequence element in the        polynucleotide with the frequencies in the variant design        sequence element frequency lookup table;    -   iv. randomly choose one codon in the polynucleotide;    -   v. randomly change the selected codon for a different codon        encoding the same amino acid;    -   vi. calculate the frequency with which each sequence element is        present in its designated frame (if any) in the modified        polynucleotide;    -   vii. compare the frequencies for each sequence element in the        polynucleotide with the frequencies in the variant design        sequence element frequency lookup table;    -   viii. if the match between the variant design sequence element        frequency lookup table and the sequence element frequencies in        the modified polynucleotide calculated in vii) is better than        the match between the variant design sequence element frequency        lookup table and the sequence element frequencies in the        original polynucleotide calculated in iii), accept the changes        from step v), else, dismiss the changes and retain original        values.

}

Output an evolved sequence, then optionally

for 10000 iterations

{

-   -   ix. calculate a value for each of the quantifiable        polynucleotide properties of interest for the polynucleotide;    -   x. compare the values calculated in viii) with the values for        the properties described in the polynucleotide sequence property        table;    -   xi. randomly choose two sequence elements from the sequence        element frequency lookup table that encode the same polypeptide        features within the polynucleotide;    -   xii. exchange the two sequence elements;    -   xiii. calculate a value for each of the quantifiable        polynucleotide properties of interest for the modified        polynucleotide;    -   xiv. compare the values calculated in xii) with the values for        the properties described in the polynucleotide sequence property        table;    -   xv. if the match between the values in the polynucleotide        sequence property table and those calculated for the modified        polynucleotide calculated in xii) is better than the match        between the polynucleotide sequence property table and those        calculated for the original polynucleotide calculated in ix),        accept the changes from step x), else, dismiss the changes and        retain original values.

}

There is evidence that in some expression systems under some conditionsthe rate of decoding of some codons can depend upon the sequence ofadjacent codons. There are some biases in codon usage dependent onneighboring codons found in the genomes of several organisms. Forexample there can be a high correlation between the 3′ terminalnucleotide of a codon and the first two nucleotides of the next codon.Thus, one quantifiable polynucleotide property is the bias of thetriplets defined by these three nucleotides (e.g., the triplets in the−1 frame register).

Decoding rate can depend highly on tRNA availability. As multiple codonsare often read by a single tRNA and multiple tRNAs can read a particularcodon, in some embodiments of the invention it can be advantageous tocluster codons according to tRNA assignments and create polynucleotidevariant sets varied by tRNA usage rather than independently varyingcodons.

Some genes contain sequence elements that affect expression at thetranscription or translation level. Making the genes less similar to theoriginal natural sequence increases the likelihood of eliminating suchelements. Varying similarity to the natural sequence also provides anadditional means to globally alter codon usage. The natural sequencerepresents a particular codon frequency bias and increasingdissimilarity will generally alter bias in a way that can differ fromother methods. Any gene sequence, not only a naturally occurringsequence, may be used as a reference for such a purpose. For example,one may wish to use a known poorly expressed variant as a reference tovary similarity to.

In some cases it has been shown to be advantageous to eliminate the useof codons in a gene that are very rarely used in the host organism.However, it can also be detrimental to reduce codon usage to only themost frequently used codons in the genome of the host. In order to findan optimal balance between eliminating slow codons maximizing the usageof the available tRNA pool, it is useful to vary the frequency cutofffor codons to be allowed in the gene design.

In some embodiments of the invention it may be useful to further modifythe codon bias in the variant set:

(i) Codon bias optimization. Results from a codon variant set (pluralityof polynucleotides) that has been synthesized and tested for one or moreexpression property can be analyzed for correlation of actual codonbias, by amino acid, to predict new bias directions that might bettercorrelate with expression, for testing in a subsequent codon variantset.

(ii) Focused codon usage tables. It may be advantageous to synthesizevariant sets where only particular codons or codon sets for particularamino acids are varied.

(iii) Specific tRNA usage. Multiple codons may be read by a single tRNAand multiple tRNAs may read the same codon in some cases. As tRNAavailability may be a critical factor in determining expression level,varying genes based on tRNA usage rather than codon usage can sometimesbe informative and yield predictive models that complement codon usagevariable sets.

(iv) 5′ codon usage. In addition to overall codon bias, the first 100bases, or the first 80 bases or the first 60 bases or the first 40 basesof the open reading frame of a polynucleotide may contain variables thatcorrelate with expression properties of the polynucleotide.

(v) Codon diversity. A measure of the overall diversity of codons usedfor each amino acid throughout a gene may be relevant to expression.

By measuring one or more expression property of polynucleotides in acodon variant set it is possible to identify the individualpolynucleotide with the best expression properties in that set. Suchdesign of a codon variant set and selection of the best expressingvariant is an aspect of the invention. This may be sufficient for adesired application.

5.5 ANALYSIS OF EXPRESSION IN CODON VARIANT SETS

It may be desirable to analyze expression data and correlate it with thecodon frequencies of the designed polynucleotides. Such analyses can beused to identify frequencies or ranges of frequencies for codons withina polynucleotide that result in superior expression properties. Theresults of this analysis can be formulated as a frequency lookup tableto be used in the design of polynucleotide sequences, or directly as anew polynucleotide sequence. The analysis of expression data from codonvariant sets to provide design rules for new polynucleotides is anaspect of the invention.

5.5.1 Models from Multivariate Regression

In one embodiment, expression data for each of the polynucleotides in acodon variant set is analyzed by determining the correlation of sequencevariables with at least one property that affects an expression propertyof polynucleotides in the codon variant set using multivariateregression. The independent variables input for regression may be, forexample, individual codon frequencies used in the plurality ofpolynucleotides in the codon variant set, estimates of tRNA usagefrequencies used, frequencies or strengths of mRNA structures,occurrence of defined sequence motifs, or summary sequence statistics,such as GC percentage, Codon Adaptiveness Index, or total length.

Several methods exist for regression of multivariate data, wherepredictive relationships between some or all of the independentvariables and expression level are determined. Examples of such methodsare Partial Least Squares (PLS) and Principal Components Regression(PCR) (Wold et al., 1993, “DNA and peptide sequences and chemicalprocesses multivariately modeled by principal component analysis andpartial least-squares projections to latent structures,” AnalyticaChimica Acta 277, 239-253). PLS algorithms, for example, seek tomaximize the correlation of the X-data (e.g., codon frequencies) andexpression while simultaneously maximizing the X-data variance capturedin the model. In doing so, the algorithm determines new orthogonalvariables, called latent variables, which are linear combinations of theoriginal variables that best capture the X-data and explain Y variation.

More generally, assessment of the contributions of relative or absolutecodon frequencies to one or more expression property can be performed byderiving a sequence-expression relationship. Such a relationship can beexpressed very generally, for example as shown in Equation AY=f(x _(AAA) , x _(AAC) , . . . x _(NNN))  (Eq A)where,

Y is a quantitative measure of an expression property (e.g., level ofprotein expressed, level of soluble protein expressed or level of activeprotein expressed),

x_(NNN) is the relative or absolute frequency of codon NNN, and

f( ) is a mathematical function that can take any of several differentforms.

In a simple embodiment of the invention, the function f can be a linearcombination of x_(i):Y=w _(AAA) x _(AAA) +w _(AAC) x _(AAC) , + . . . w _(NNN) x _(NNN)  (Eq.b)where,

w_(NNN) is a weight (or coefficient of x_(NNN)).

In some embodiments, to derive a sequence-expression relationship, a setof descriptors (x_(NNN)) that can describe the frequencies of some orall of the codons within the codon variant set is identified. Values ofY for each member of the codon variant set are measured. Values for eachweight (w_(NNN)) are then calculated such that the differences betweenvalues predicted for each value of Y by Equation A and those observedexperimentally are minimized for the codon variant set, or for aselected subset of such codon variants.

The minimization step above can also use weights for differentexpression property predictions and, in general, can use a lossfunction. In one embodiment this loss function can be squared errorloss, where weights that minimize the sum of squares of the differencesbetween predicted and measured values for the dataset are computed.

In some embodiments statistical regression methods are used to identifyrelationships between dependent (x_(NNN)) and independent (Y) variables.Such techniques include, but are not limited to, linear regression,non-linear regression, logistic regression, multivariate data analysis,and partial least squares regression. See, for example, Hastie, TheElements of Statistical Learning, 2001, Springer, N.Y.; Smith,Statistical Reasoning, 1985, Allyn and Bacon, Boston. In one embodiment,regression techniques like the PLS (Partial Least Square) can be used tosolve for the weights (w_(NNN)) in the equation X. Partial Least Squares(PLS) is a tool for modeling linear relationships between descriptors.The method is used to compress the data matrix composed of descriptors(variables) of variant sequences being modeled into a set of latentvariables called factors. The number of latent variables is much smallerthan the number of variables (descriptors) in the input sequence data.For example, if the number of input variables is 100, the number oflatent variables can be less than 10. In some embodiments, the factorsare determined using the nonlinear iterative partial least squaresalgorithm. The orthogonal factor scores are used to fit a set ofactivities to the dependent variables. Even when the predictors arehighly collinear or linearly dependent, the method finds a good model.Alternative PLS algorithms like the SIMPLS can also be used forregression. In such methods, the contribution to the activities fromevery variable can be deconvoluted to study the effect of sequence onthe function of the protein.

In some embodiments, modeling techniques are used to derivesequence-expression relationships. Such modeling techniques includelinear and non-linear approaches. Linear and non-linear approaches aredifferentiated from each other based on the algebraic relationships usedbetween variables and responses in such approaches. In the system beingmodeled, the input data (e.g., variables that serve as descriptors ofthe biopolymer sequence), in turn, can be linearly related to thevariables provided or non-linear combinations of the variables. It istherefore possible to perform different combinations of models anddata-types: linear input variables can be incorporated into a linearmodel, non-linear input variables can be incorporated into a linearmodel and non-linear variables can be incorporated into a non-linearmodels.

Many functional forms of f( ) (Eqn. A) can be used and the functionalform can be combined using weights defined for analysis. For example,Function f( ) can assume a non-linear form. An example of non-linearfunctional form is:Y=w ₁₂ *x ₁ *x ₂ +w ₁₃ *x ₁ *x ₃ + . . . w _(nn) *x _(n) *x _(n)Non-linear functions can also be derived using modeling techniques suchas machine learning methods. For example, the codon (x_(NNN))-expression(Y) data to predict the activities of any sequence given the descriptorsfor a sequence can be determined using neural networks, Bayesian models,generalized additive models, support vector machines and classificationusing regression trees.

In some embodiments, supervised learning techniques are used to identifyrelationships between relative or absolute codon frequencies in thedesigned set and measured expression properties. Such supervisedlearning techniques include, but are not limited to, Bayesian modeling,nonparametric techniques (e.g., Parzen windows, k_(n)-Nearest-Neighboralgorithms, and fuzzy classification), neural networks (e.g., hopfieldnetwork, multilayer neural networks and support vector machines), andmachine learning algorithms (e.g., algorithm-independent machinelearning). See, for example, Duda et al., Pattern Classification, 2^(nd)edition, 2001, John Wiley & Sons, Inc. New York; and Pearl,Probabilistic Reasoning in Intelligent Systems: Networks of plausibleInference, Revised Second Printing, 1988, Morgan Kaufmann, SanFrancisco. For example, the codon (x_(NNN))-expression (Y) data can beused to predict the expression property of any sequence given the codondescriptors for a sequence using a neural network. The input for thenetwork is the descriptors and the output is the predicted value of Y.The weights and the activation function can be trained using superviseddecision based learning rules. The learning is performed on a subset ofvariants called the training set and performance of the network isevaluated on a test set.

In some embodiments, unsupervised learning techniques are used toidentify relationships between relative or absolute codon frequencies inthe designed set and measured expression properties. Such unsupervisedlearning techniques include, but are not limited to stochastic searches(e.g., simulated annealing, Boltzmann learning, evolutionary methods,principal component analysis, and clustering methods). See, for example,Duda et al., Pattern Classification, 2^(nd) edition, 2001, John Wiley &Sons, Inc. New York. For example, the weights in equation B can beadjusted by using monte carlo and genetic algorithms. The optimizationof weights for non-linear functions can be complicated and no simpleanalytical method can provide a good solution in closed form. Geneticalgorithms have been successfully used in search spaces of suchmagnitude. Genetic algorithms and genetic programming techniques canalso be used to optimize the function form to best fit the data. Forinstance, many recombinations of functional forms applied on descriptorsof the sequence variants can be applied.

In some embodiments, boosting techniques are used to construct and/orimprove models developed using any of the other techniques describedherein. A model of the sequence-expression relationship can be describedas a functional form whose parameters have been trained for the inputdata (Y and x_(NNN)). Many algorithms/techniques to build models havebeen described. Algorithms applied on a specific dataset can be weak inthat the predictions can be less accurate or “weak” (yielding poormodels). Models can be improved using boosting techniques. See, forexample, Hastie et al., The Elements of Statistical Learning, 2001,Springer, N.Y. The purpose of boosting is to combine the outputs of many“weak” predictors into a powerful “committee.” In one embodiment of theinvention, boosting is applied using the AdaBoost algorithm. Here, theprediction algorithm is sequentially applied to repeatedly modifiedversions of the data thereby producing a sequence of models. Thepredictions from all of these models are combined through a weightedmajority vote to produce the final prediction. The data modification ateach step consists of applying weights (W^(b) _(i)) to each of the itraining observations. Initially weights are set to 1/N, where N is thenumber of training observation (sequence-activity data). The weights aremodified individually in each successive iteration. Trainingobservations that were predicted poorly by a particular model have theirweights increased and training observations that were predicted moreaccurately have their weights decreased. This forces each successivemodel to concentrate on those training observations that are issued bythe previous model. The step of combining the models to produce a“committee” assigns a weight to each model based on the overallprediction error of that model.

The various modeling techniques and algorithms described herein can beadapted to derive relationships between one or more expressionproperties or functions of a nucleic acid sequence of polynucleotide andtherefore to make multiple predictions from the same model. Modelingtechniques that have been adapted to derive sequence-expressionrelationships for polynucleotides are within the scope of the presentinvention. Some of these methods derive linear relationships (forexample partial least squares projection to latent structures) andothers derive non-linear relationships (for example neural networks).Algorithms that are specialized for mining associations in the data arealso useful for designing sequences to be used in the next iteration ofsequence space exploration. These modeling techniques can robustly dealwith experimental noise in the activity measured for each variant. Oftenexperiments are performed in replicates and for each variant there willbe multiple measurement of the same activity. These multiplemeasurements (replicate values) can be averaged and treated as a singlenumber for every variant while modeling the sequence-expressionrelationship. The average can be a simple mean or another form of anaverage such as a geometric or a harmonic mean. In the case of multiplemeasurements, outliers can be eliminated. In addition, the errorestimation for a model derived using any algorithm disclosed herein canincorporate the multiple measurements through calculating the standarddeviation of the measurement and comparing the predicted activity fromthe model with the average and estimate the confidence interval withinwhich the prediction lies. Weights for observations to be used in modelscan also be derived from the accuracy of measurement, for example,through estimating standard deviation and confidence intervals. Thisprocedure can put less emphasis on variants whose measurements are notaccurate. Alternatively, these replicate values can be treatedindependently. This will result in duplicating the sequences in thedataset. For example, if sequence variant, represented by descriptorvalues {x_(j)}^(i1), has been measured in triplicates (Y_(i1), Y_(i2),Y_(i3)), the training set for modeling will include descriptor value{x_(j)}^(i2) with activity Y_(i2) and {x_(j)}^(i3) with activity Y_(i3)in addition to {x_(j)}^(i1) with activity Y_(i1), where{x_(j)}^(i1)={x_(j)}^(i2)={x_(j)}^(i3).

A representative modeling routine in accordance with one embodiment ofthe invention comprises the following steps.

Step 302. Relevant descriptors of the sequence are identified. Thesedescriptors can convey absolute or relative frequencies of sequenceelements including codons. They can also contain information about thelocation of the sequence element within the polynucleotide, or thedistribution of the sequence element throughout the polynucleotide.

In some embodiments, it is advantageous to identify absolute or relativecodon frequencies based on factors including, but not limited toabsolute and relative positions within the polynucleotide. For example,a weight of a can be assigned for variables in domain A of thepolynucleotide and a weight of b can be assigned for variables in domainB of the polynucleotide. This weighting can also incorporate constraintsand other functional considerations that may or may not be measured inexperiments, but which can be fully or partially be predicted usingcomputational techniques.

Step 304. In step 304 the parameters for the functional form of thesequence-activity relationship are optimized to obtain a model byminimizing the difference between the predicted values and real(measured) values of the expression property of the polynucleotide. Suchoptimization adjusts the individual weights for each descriptoridentified in preceding steps using a refinement algorithm such as leastsquares regression techniques. Other methods that use alternative lossfunctions for minimization can be used to analyze any particulardataset. For example, in some polynucleotide sequence-expression datasets, the expression properties may not be distributed evenly throughoutthe measured range. This will skew the model towards data points in theactivity space that are clustered. This can be disadvantageous becausedatasets often contain more data for polynucleotide variants with lowlevels of activity, so the model or map will be biased towards accuracyfor these polynucleotides which are of lower interest. This skeweddistribution can be compensated for by modeling using a probabilityfactor or a cost function based on expert knowledge. This function canbe modeled for the activity value or can be used to assign weights todata points based on their activity. As an example, for a set ofactivities in the range of 0 to 10, transforming the data with asigmoidal function centered at five will give more weight to sequenceswith activity above five. Such a function can optionally also be alteredwith subsequent iterations, thereby focusing the modeling on the part ofthe dataset with the most desired functional characteristics. Thisapproach can also be coupled with exploring techniques like a Tabusearch, where undesired space is explored with lower probabilities.

In some embodiments, algorithms that optimizes the sequence-expressionmodel for the dataset by randomly starting with a solution (e.g.,randomly assigning weights w_(NNN)) and using methods like hill-descentand/or monte-carlo and/or genetic algorithm approaches to identifyoptimal solutions.

In embodiments directed to codon optimization, robustness of the modelsused is a significant criterion. Thus, obtaining several sub-optimalsolutions from various initial conditions and looking at all the modelsfor common features can be a desirable methodology for ensuring therobustness of the solution. Another way to obtain robust solutions is tocreate bootstrap data sets based on the input data, than estimate ap-value or confidence on the various coefficients of the model. Inaddition boosting techniques like AdaBoost can be used to obtain a“committee” based solution.

Step 306. Many mathematical modeling techniques for deriving asequence-expression correlation are evaluated. Preferred mathematicalmodeling techniques used to identify and capture the sequence-activitycorrelation handle (i) very large numbers of variables (e.g. 20 or more)and correlations between variables, (ii) linear and non-linearinteractions between variables, and (iii) are able to extract thevariables responsible for a given functional perturbation for subsequenttesting of the mathematical model (e.g., models should be easilyde-convoluted to assign the effect of variables describing the aminoacids substitution with activities).

Step 308. In step 308 the coefficients (parameters) of the model(s) aredeconvoluted to see which codon frequencies (variables/descriptors ofthe variants) influence the expression properties of the polynucleotide.It can be important to identify which descriptor(s) of thepolynucleotide are important for the activity of interest. Some of thetechniques, such as partial least squares regression (SIMPLS) that usesprojection to latent structures (compression of data matrix intoorthogonal factors) may be good at directly addressing this pointbecause contributions of variables to any particular latent factors canbe directly calculated. See, for example, Bucht et al., 1999, BiochimBiophys Acta. 1431:471-82; and Norinder et al., 1997, J Pept Res 49:155-62. Other methods such as neural networks can learn from the datavery well and make predictions about the activity of entirepolynucleotides, but it may be difficult to extract information, such asindividual contributing features of the polynucleotide from the model.Modeling techniques/methods that directly correlate the codon frequencyvariations to the expression property are preferred because they can beused to derive the sequence-expression map (relationship) to constructnew polynucleotides or codon bias tables not in dataset that havepreferentially higher activities. These methods can be adapted toprovide a direct answer and output in desired forms.

Step 310. In step 310 the models developed using various algorithms andmethods in the previous step can be evaluated by cross validationmethods. For example, by randomly leaving data out to build a model andmaking predictions of data not incorporated into the model is a standardtechnique for cross validation. In some instances of polynucleotideexpression engineering, data may be generated over a period of months.The data can be added incrementally to the modeling procedure as andwhen such data becomes available. This can allow for validation of themodel with partial or additional datasets, as well as predictions forthe properties of biopolymer sequences for which activities are stillnot available. This information may then be used to validate the model.

In one embodiment of the present invention, average values and standarddeviations for weight functions can be obtained by omitting a part ofthe available data. Either individual sequences and their associatedexpression activities or individual codons can be left out. Asequence-expression relationship can then be constructed from thispartial data. This process can be repeated many times, each time thedata to leave out is selected randomly. Finally an average and range ofvalues for each weight function is calculated. The weight functions canthen also be ranked in order of their importance to activity. The rangeof values for each weight can provide a measure of the confidence withwhich the weight is assigned. It can also provide a measure of theimportance of the variable in determining the expression property. Forexample in some instances, the larger the standard deviation for avariable weight, the larger the range of values for that variable thatare associated with desirable expression properties.

To assess the probability that a codon frequency is associated with anactivity by random chance, the same weight function calculations can beperformed when the sequences and activities are randomly associated. Inthis case there should be no relationship between codon frequencies andexpression property, so weight functions arise only by chance. A measureof the confidence for the weight function can then be calculated. It isrelated to the number of standard deviations by which the valuecalculated when sequences and activities are correctly associatedexceeds the value calculated when they are randomly associated. Theabove methods on model assessment, model inference and averaging arediscussed in detail by Hastie et al., 2001, Springer Verlag, series instatistics.

Step 312. In step 312 new polynucleotide sequences or codon biases thatare predicted to possess one or more desired property are derived.Alternatively it can be desirable to rank order the input variables fordetailed sequence-expression correlation measures. The model can be usedto propose codon biases that have high probabilities of being improved.Polynucleotides designed to conform to such codon biases can thenoptionally be synthesized and tested. In one embodiment, this can beachieved if the effects of various sequence features of thepolynucleotides on their functions are known based on the modeling.Alternatively, for methods like neural networks, 10³ or 10⁶ or 10⁹ or10¹² or 10¹⁵ or 10¹⁸ or as many as 10⁸⁰ sequences can be evaluated insilico. Then those predicted by the model to possess one or more desiredproperties are selected.

Step 314. The statistical quality of the model fit to the input data isevaluated in step 314. Validation of sequence-expression correlation canbe internal, using cross-validation of the data, or preferably external,by forecasting the functional perturbation of a set of new sequencesderived from the model. Sequences with predicted values of theirfunctional perturbations are then physically made and tested in the sameexperimental system used to quantify the training set. If thesequence-expression relationship of the dataset is satisfactorilyquantified using internal and external validation, the model can beapplied to a) predict the functional value of other related sequencesnot present in the training set, and b) design new sequences within thedescribed space that are likely to have a function value that is outsideor within the range of function given by the training set.

The initial set of data can be small, so models built from it can beinaccurate. Improving the modeled relationship further depends uponobtaining better values for weights whose confidence scores are low. Toobtain this data, additional variants designed will provide additionaldata useful in establishing more precise sequence-expressionrelationships.

The output from each method for modeling a sequence-activityrelationship can be one or more of: (i) a regression coefficient, weightor other value describing the relative or absolute contribution of eachcodon frequency or combination of codon frequencies to one or moreexpression activity of the polynucleotide, (ii) a standard deviation,variance or other measure of the confidence with which the valuedescribing the contribution of the codon frequency or combination ofcodon frequencies to one or more expression activity of thepolynucleotide can be assigned, (iii) a rank order of preferred codons,(iv) the additive & non-additive components of each codon frequency orcombination of codon frequencies, (v) a mathematical model that can beused for analysis and prediction of the functions of in silico generatedsequences. Such output can be used to generate a frequency lookup table.For example, codons that are assigned more significant weights in themodeling described above can be assigned frequency ranges in thefrequency lookup table that contain higher frequencies than codons thatare assigned less significant weights.

5.5.2 Codon Frequency Data

One preferred way to define independent variables for sequence variantsis as individual codon frequencies. As one example, the mean-centeredfrequency occurrence of each of the 61 sense codons for a set of N genesis input as the independent variable matrix (N×61X-data matrix) and theN absolute or relative expression levels are input as the dependentvariables. PLS modeling is used to define useful latent variables fromthe codon frequencies for prediction of expression. The number of latentvariables to include in the regression is chosen such that a maximumcapture of the codon and expression data is achieved without overfitting. This is achieved using cross-validation methods to assess theability of models based on subsets of the data to predict expression ofthe excluded sets. Latent variables are included only if theysignificantly reduce the cross-validation error. The regression returnsseveral statistics that express the relationship of the codonfrequencies to the latent variables (e.g., the variable loadings on thelatent variables) and to the expression levels (e.g., the regressionvectors for the Y data). The regression vector for Y is a matrix ofvalues for each of the codon frequencies that expresses the relativecontribution of each codon to the predicted expression level, accordingtoY _(M) =R(X _(M))where R is the regression vector, Y_(M) is the predicted mean centeredexpression level, and X_(M) is the N×1 mean centered codon frequencymatrix for a given gene. Thus the regression vector matrix, R, may beused to predict expression of any given gene based on the codonsfrequencies used for that gene.

5.5.3 Variable Selection Methods

Additional analyses of the data may be used to further refine models andto indicate the relative significance of the codon frequencies to theexpression model. One useful statistic is the Variable Importance in theProjection (VIP) which is a measure of the dependence of the model fiton inclusion of a variable. VIP values greater than one are generallyconsidered significant. Analysis of the VIP can be used to pick whichcodons are most likely important for influencing expression. Anothermethod of variable selection involves the use of a Genetic Algorithm(GA). In a typical GA run for codon frequency analysis, ˜100 randomsubsets, or “samples”, of 15-25 of the 61 sense codons are generated andevaluated for their ability to explain the data in PLS modeling. Theerror of the PLS fit of the data in cross validation (RMSECV) is used todistinguish the subsets. Those that yield lower than median RMSECV areretained. The codon sets used by random pairs of these selected samplesare then recombined at two random crossover points to create new progenysamples. The resulting samples, the original selected and their progeny,are then analyzed for fit as before and the best half are used to createthe next sample generation. At each generation, mutation (substitutingone codon variable in a sample for another) is allowed to prevent themodel from prematurely eliminating or fixing under- or overrepresentedvariables, respectively. The entire process is repeated until there isconvergence in makeup and performance of the selected population. Thefinal samples will have codon sets that better fit the expression data.

For variable selection it is useful to analyze the frequency ofinclusion of codons used in the sample population as the GA iterates.Codons that are most enriched by the GA are taken as most likelysignificant for expression. One may also use the best sample codon setsgenerated by the GA as refined, potentially more precise, sets formodeling expression. The codons whose frequencies have most effect onexpression can also be determined using other combinations of analyticalmethod such as dimension-compressing techniques including partial leastsquares regression and principal component analysis, with stochasticsearch methods including genetic algorithms, monte carlo algorithms orsimulated annealing algorithms.

5.5.4 Additional Methods for Determining an Expression Property ofPolynucleotides in an Expression System

Another aspect provides a method of determining at least one propertythat affects an expression property of polynucleotides in an expressionsystem. The method comprises constructing a first plurality ofpolynucleotides. The first plurality of polynucleotides comprises fiveor more polynucleotides, ten or more polynucleotides, twenty or morepolynucleotides, thirty or more polynucleotides, or more than 50polynucleotides. Each polynucleotide in the first plurality ofpolynucleotides encodes a predetermined polypeptide sequence. Afrequency with which a first sequence element is used in a firstpolynucleotide in the first plurality of polynucleotides is differentthan a frequency with which the first sequence element is used in asecond polynucleotide in the first plurality of polynucleotides. Eachrespective polynucleotide in the first plurality of polynucleotidesindividually in the expression system. An expression property value ofeach respective polynucleotide in the first plurality of polynucleotidesin the expression system is measured, thereby constructing a dataset.The dataset comprises, for each respective polynucleotide in the firstplurality of polynucleotides, a representation of sequence elementoccurrence in the respective polynucleotide and the measured expressionproperty value of the respective polynucleotide.

A sequence element is a defined sequence of nucleotides and may alsoinclude a reading frame designation relative to the reading frame of apolypeptide encoded by a polynucleotide. Sequence elements include butare not limited to codons, nucleotide triplets in the +3 reading frame(encoding the “wobble” or third base of one codon and the first twobases of the following codon) and nucleotide hexamers in the +1 readingframe (encoding an adjacent pair of codons or “codon pair”). Otherquantifiable sequence properties include GC content, mRNA secondarystructures in particular regions of an mRNA produced by thepolynucleotide for example covering the start codon, the degree ofsequence identity to a reference sequence, the presence of ribosomebinding sites, polyadenylation signals, polynucleotide splice signals,the annealing temperature for a sub-sequence of predetermined lengthwithin the polynucleotide for any other sub-sequence within thepolynucleotide, repeated sequence elements or homopolymer stretches.Variation in GC %, either of the entire codon or simply at one position(e.g., the more variable 3′ terminal nucleotide) is a useful way to varyoverall bias and can add diversity in design. These properties can betabulated in variant design polynucleotide sequence property lookuptables that can be used in a similar way to sequence element frequencylookup tables: a polynucleotide can be designed such that it conforms tothe values for quantifiable sequence properties described in one of thepolynucleotide sequence property tables.

In the method a final model is computed. The final model comprises (i) aplurality of variables, each variable in the plurality of variablesdescribing a frequency of occurrence of one or more sequence elements inthe first plurality of polynucleotides, or another quantifiable sequenceproperty and (ii) a plurality of weights, each weight in the pluralityof weights corresponding to one or more variables in the plurality ofvariables. In some embodiments, the plurality of variables is three ormore variables, four or more variables, five or more variables, six ormore variables, between five and twenty-five variables, between four andsixty variables, or less than forty variables. In some embodiments, theplurality of weights is three or more weights, four or more weights,five or more weights, six or more weights, between five and twenty-fiveweights, between four and sixty weights, or less than forty weights. Insome embodiments, there is one-to-one correspondence between eachvariable in the plurality of variables and each weight in the pluralityof weights. In some embodiments the final model describes a variation inthe measured expression property values of the first plurality ofpolynucleotides from the measuring as a function of the plurality ofvariables and their corresponding weights.

From the final model at least one property that affects an expressionproperty of polynucleotides in the expression system is determined. Theat least one property is an effect that the frequency of occurrence ofone or more sequence elements has on the expression property ofpolynucleotides in the expression system.

In some embodiments, computation of the final model comprises searchinga multivariate space using a global optimization algorithm therebycomputing the final model. This multivariate space comprises theplurality of variables. In other words, this multivariate spacecomprises all possible combinations of all possible values for eachvariable in the plurality of variables. For instance, consider thehypothetical case where the plurality of variables consists of variablesA and B, and that each variable can have the value 1 or 2. In this case,the multivariate space that comprises the plurality of variables is{(A1, B1), (A1, B2), (A2, B1}, and (A2, B2)), wherein (A1, B1) meansthat variable A has value 1 and variable B has value 1, and so forth. Infact, there are typically many variables (e.g., between five and fiftyvariables) in the plurality of variables and each variable can adopt anyof several different values. Further, each variable is weighted by oneor more weights. Thus, the multivariate space is very large. Because ofthe large size of the multivariate space, a global optimizationalgorithm is used in some embodiments to help find the final model inthe multivariate space. In many embodiments, there is no guarantee thatthe final model is the absolute best model for the dataset. It is simplythe best model found by the global optimization algorithm given theinput dataset.

The global optimization algorithm assigns a respective score to eachrespective test model in a plurality of test models in order to find thefinal model. Each of these test models is some combination of theplurality of variables and the plurality of weights. For example, if theplurality of variables consists of fifty variables, a given test modelmay consist of fifteen variables and fifteen corresponding weights,where each respective weight is for a corresponding variable. Thus,there is no requirement that each test model include each variable inthe plurality of variables. In fact, in the case where variablesrepresent codon frequency, it is desirable to limit the number ofvariables so that the test model imposes the fewest possiblerestrictions on codon choice.

Models that use fewer variables to explain the variance in measuredexpression properties of the plurality of polynucleotides are preferredbecause they impose less restrictions on codon choice relative to modelsthat include more variables representing codon choice. For example,consider a first test model that includes forty variables, where each ofthe variables represents a different codon in the set of naturallyoccurring codons, and explains seventy percent of the variation in themeasured expression property of the plurality of polynucleotides and asecond test model that includes twenty variables, where each of thevariables represents a different codon in the set of naturally occurringcodons, and explains sixty-nine percent of the variation in the measuredexpression property of the plurality of polynucleotides. In this case,the second test model is preferred to the first test model because thesecond test model imposes restrictions on only twenty different codonswhereas the first model imposes restrictions on forty different codons.Thus, if the first model were used to design a polynucleotide sequence,there would be limitations on the frequency with which forty differentcodons could be used in the polynucleotide. This would reduce the numberof possible locations where other sequence elements, such asrestrictions sites, could be located in the designed polynucleotidesequence.

The global optimization algorithm assigns a respective score to eachrespective test model in a plurality of test models in order to find thefinal model. Each respective score is a quantification of an agreementbetween (i) expression property values calculated for each respectivepolynucleotide in the first plurality of polynucleotides by a test modelin the plurality of test models and (ii) expression property valuesmeasured for each respective polynucleotide in the first plurality ofpolynucleotides by the measuring described above. In some embodiments,this agreement is a correlation or some other similarity metric. In someembodiments, each test model in the plurality of test models comprises anumber of variables in the plurality of variables and a correspondingnumber of weights in the plurality of weights.

In some embodiments, the plurality of test models is ten or more models,one hundred or more models, one thousand or more models, ten thousand ormore models, or one hundred thousand or more models.

In some embodiments, the one or more sequence elements are each codonsthat are in the same reading frame with respect to the predeterminedpolypeptide sequence encoded by a polynucleotide in the plurality ofpolynucleotides. In some embodiments, a sequence element in the one ormore sequence elements is a codon pair that is in the same reading framewith respect to the predetermined polypeptide sequence encoded by apolynucleotide in the first plurality of polynucleotides. In someembodiments, a sequence element in the one or more sequence elements isa nucleotide triplet in the +3 reading frame (encoding the “wobble” orthird base of one codon and the first two bases of the following codon).

In some embodiments, the computation of the final model furthercomprises computing a confidence of each respective test model in theplurality of test models and using the respective score for a respectivetest model in the plurality of test models and the confidence for therespective test model to determine whether to accept or reject therespective test model as the final model.

In some embodiments, the global optimization algorithm is a heuristicsearch comprising a plurality of steps where, at each step in theplurality of steps, the global optimization algorithm comprises (i)determining a respective score for each respective test model in asubset of the plurality of test models, (ii) enriching the plurality oftest models by removing a portion of the subset of the plurality of testmodels from the plurality of test models based on respective scoresassigned to the test models in the subset of test models, and (iii)enriching the plurality of test models by adding a new subset of testmodels to the plurality of test models, where each test model in the newsubset of test models is derived from values for weights in one or moretest models already in the plurality of test models. An example of howsuch an embodiment can be carried out is a genetic algorithm. First, instep (i) a respective score for each respective test model in a subsetof the plurality of test models is computed. For example, one thousandmodels are computed. Each respective model in the one thousand testmodels is scored for an agreement between (i) expression propertiescalculated for each respective polynucleotide in the first plurality ofpolynucleotides by the test model and (ii) expression propertiesmeasured for each respective polynucleotide in the first plurality ofpolynucleotides for which measurement data is available. Next, in (ii)some of the test modes are removed from the plurality (from the set of1000 test models in this example) of test models. Next, in (iii) theplurality of test models is enriched by adding more test models. Thetest models added in (iii) are derived from the test models that werenot thrown out in (ii). A test model can be derived from other models bytaking some of the variables and weights from the other models to form anew combination in the manner known in genetic algorithms. In this way,the global optimization algorithm can efficiently converge on a suitablefinal model.

In some embodiments, the global optimization algorithm refines a testmodel in the plurality of test models, before assigning a score to thetest model, by principal component analysis in which the variables inthe test model are reduced to two or more latent variables that accountfor all or a portion of a variation in the measured expression propertyvalues of the first plurality of polynucleotides. Principal componentanalysis is described in Hastie, 2003, The Elements of StatisticalLearning, Springer, N.Y., pp. 485-493 which is hereby incorporated byreference herein for such purpose. In some embodiments, the globaloptimization algorithm is a heuristic search. Examples of heuristicsearches include, but are not limited to, an evolutionary algorithm, aswarm-based optimization algorithm, a memetic algorithm, or adifferential evolution algorithm.

In some embodiments, each of the one or more sequence elements in thefinal model is defined by a sequence of nucleotides and a reading framerelative to the predetermined polypeptide sequence encoded by apolynucleotide in the plurality of polynucleotides.

In some embodiments, the global optimization algorithm is a stochasticsearch. Examples of stochastic searches include, but are not limited to,a simulated annealing algorithm, a directed monte-carlo samplingalgorithm, a stochastic tunneling algorithm, a parallel temperingalgorithm, a monte-carlo with minimization algorithm or a continuationmethod. See for example, Horst et al., 2000, Introduction to GlobalOptimization, Second Edition, Kluwer Academic Publishers; Neumaier,2004, Complete Search in Continuous Global Optimization and ConstraintSatisfaction, pp. 271-369 in: Acta Numerica 2004 (A. Iserles, ed.),Cambridge University Press; Mongeau et al., 2000, Optimization Methods &Software 13(3), pp. 203-226; Kirkpatrick et al., 1983, Science220:671-680, Hamacher, 2006, Europhys. Lett. 74(6):944, 2006; Hamacherand Wenzel, 1999, Landscape. Phys. Rev. E, 59(1):938-941; Wenzel andHamacher, 1999, Phys. Rev. Lett., 82(15):3003-3007; Hansmann, 1997,Chem. Phys. Lett., 281:140; Zhijun, November 1996, “The effective energytransformation scheme as a special continuation approach to globaloptimization with application to molecular conformation,” TechnicalReport, Argonne National Lab., IL (United States), November 1996, eachof which is hereby incorporated by reference herein in its entirety.

In some embodiments, the expression property value of a respectivepolynucleotide in the first plurality of polynucleotides in theexpression system is (i) a total amount of protein encoded by therespective polynucleotide that is expressed in the expression system ina defined time, (ii) a total amount of active protein encoded by therespective polynucleotide that is expressed in the expression system ina defined time, or (iii) a total amount of soluble protein encoded bythe respective polynucleotide that is expressed in the expression systemin a defined time.

In some embodiments, the relative frequency of each respectivesynonymous codon in a plurality of synonymous codons for each of two ormore, three or more, four or more, five or more, six or more, seven ormore, eight or more, nine or more, or ten or more amino acids encoded bya reading frame of two or more, three or more, four or more, five ormore, six or more, seven or more, eight or more, nine or more, ten ormore, twenty or more or one hundred or more polynucleotides in theplurality of polynucleotides is varied in the two or more, three ormore, four or more, five or more, six or more, seven or more, eight ormore, nine or more, ten or more, twenty or more or one hundred or morepolynucleotides.

In some embodiments, the constructing comprises encoding a firstpolynucleotide in the first plurality of polynucleotides using a firstfrequency lookup table, where the first frequency lookup table specifiesa first target frequency range for the use of a first sequence elementin a polynucleotide and where a first frequency that the first sequenceelement is used in the first polynucleotide is within the first targetfrequency range Further, in such embodiments, the constructing comprisesencoding a second polynucleotide in the first plurality ofpolynucleotides using a second frequency lookup table, where the secondfrequency lookup table specifies a second target frequency range for theuse of the first sequence element in a polynucleotide and where a secondfrequency that the first codon is used in the second polynucleotide iswithin the second target frequency range. Here, the first frequencyrange is different than the second frequency range.

In some embodiments, each test model in a first subset of test models inthe plurality of test models each consist of a first number of variablesin the plurality of variables, each test model in a second subset oftest models in the plurality of test models each consist of a secondnumber of variables in the plurality of variables and the first numberis different than the second number.

In some embodiments, the plurality of variables comprises a variable foreach of two or more, three or more, four or more, five or more, six ormore, seven or more, eight or more, nine or more, or ten or morenaturally occurring codons. In some embodiments, each polynucleotide inthe first plurality of polynucleotides encodes the same amino acidsequence.

In some embodiments, the first plurality of polynucleotides comprises aplurality of subsets of polynucleotides and each respective subset ofpolynucleotides in the plurality of subsets polynucleotides encodes adifferent amino acid sequence, and each polynucleotide in a subset ofpolynucleotides in the plurality of polynucleotides encodes the sameamino acid sequence.

In some embodiments, the method further comprises constructing a secondplurality of polynucleotides, where a frequency that a sequence elementis used in the second plurality of polynucleotides is determined by theat least one property from the determining.

In some embodiments, the method further comprises constructing afrequency lookup table for the expression system from the final model,where the frequency lookup table provides a sequence element frequencyrange for each of two or more sequence elements, three or more sequenceelements, four or more sequence elements, five or more sequenceelements, six or more sequence elements, or seven or more sequenceelements.

5.5.5 Use of Principal Component Analysis for Determining an ExpressionProperty of Polynucleotides in an Expression System

In some embodiments, principal component analysis is used to determine aproperty that affects an expression property of polynucleotides in anexpression system. Such an approach is advantageous because it preventsoverfitting of the measured expression data using too many variables.

One aspect provides a method of constructing a frequency lookup tablefor an expression system, where the frequency lookup table comprises aplurality of codons. In some embodiments, the plurality of codons isfive or more codons, ten or more codons, fifteen or more codons, twentyor more codons, twenty-five or more codons, between five and ten codons,or less than fifty codons. The method comprises constructing a pluralityof polynucleotides, each polynucleotide in the plurality ofpolynucleotides encoding a predetermined polypeptide sequence, where afrequency with which a first codon is used in a first polynucleotide inthe plurality of polynucleotides, relative to the frequency with whichall other codons in the plurality of codons that are synonymous to thefirst codon are used in the first polynucleotide, is different than afrequency with which the first codon is used in a second polynucleotidein the plurality of polynucleotides, relative to the frequency withwhich all other codons in the plurality of codons that are synonymous tothe first codon are used in the second polynucleotide. In someembodiments, the plurality of nucleotides comprises five or morepolynucleotides, ten or more polynucleotides, twenty or morepolynucleotides, or one hundred or more polynucleotides.

In the method, each respective polynucleotide in the plurality ofpolynucleotides is expressed individually in an expression system. Forexample, if the expression system is E. Coli, each respectivepolynucleotide is introduced into a sample of E. Coli (e.g., in anexpression vector that is transfected into the sample of E. Coli) andexpressed. Then, an expression property value of each respectivepolynucleotide in the plurality of polynucleotides in the expressionsystem is measured thereby constructing a dataset. The datasetcomprises, for each respective polynucleotide in the plurality ofpolynucleotides, a representation of codon occurrence in the respectivepolynucleotide and the measured expression property value of therespective polynucleotide. In some embodiments, the representation ofcodon occurrence is relative to other synonymous codons in thepolynucleotide. For example, consider the case where there are twosynonymous codons, termed codon A and codon B. The representation ofcodon occurrence in the dataset would comprise the relative frequencythat codon A is used relative to codon B. In some embodiments, therepresentation of codon occurrence is relative to all other codons inthe polynucleotide. For example, consider the case where there arethirteen different codons used in the polynucleotide. The representationof codon occurrence for any one of these codons would be relative to thecollective codon occurrence of all thirteen codons, which would be thesame as the number of times the codon was used divided by the totalnumber of amino acids in the encoded polypeptide sequence. Otherrepresentations of codon occurrence are possible. For example, thenumber of times a codon was used in the polynucleotide could be storedin the dataset.

Next in the method a model is computed. The model comprises (i) aplurality of variables, each variable in the plurality of variablesdescribing a frequency of occurrence of one or more codons in the firstplurality of polynucleotides and (ii) a plurality of weights, eachweight in the plurality of weights corresponding to one or morevariables in the plurality of variables. In some embodiments, theplurality of variables consists of two or more variables, three or morevariables, four or more variables, five or more variables, six or morevariables, ten or more variables, twenty or more variables, between fiveand one hundred variables, or less then forty variables. In someembodiments, the plurality of weights consists of two or more weights,three or more weights, four or more weights, five or more weights, sixor more weights, ten or more weights, twenty or more weights, betweenfive and one hundred weights, or less then forty weights. The modeldescribes a variation in the measured expression property values of theplurality of polynucleotides from the measuring as a function of theplurality of variables and their corresponding weights. In someembodiments, each variable is for the occurrence of a particular codonin the set of naturally occurring codons used in a polynucleotide and aweight for the variable is a significance on this codon on determiningan expression property of the polynucleotide. In some embodiments, eachweight in the plurality of weights is for a codon in the plurality ofcodons.

The method further includes refining the model by principal componentanalysis in which the plurality of variables in the model are reduced toa plurality of latent variables, including a first latent variable,where the plurality of latent variables collectively account for all ora portion of a variation in the measured expression property values ofthe plurality of polynucleotides from the measuring, and where eachcodon in the plurality of codons has a corresponding load in the firstlatent variable.

The method further includes constructing the frequency lookup table forthe expression system from the final model, where the frequency lookuptable provides a corresponding codon frequency range for each of two ormore codons in the plurality of codons, and where the codon frequencyrange for a codon in the frequency lookup table is determined by thecorresponding load of the codon in the first latent variable.

The method further comprises outputting the frequency lookup table to auser interface device, a tangible computer readable storage medium; ordisplaying the frequency lookup table in user readable form.

In some embodiments, each frequency in a frequency range for arespective codon in the frequency lookup table is a frequency that therespective codon can be incorporated into a polynucleotide relative tothe frequencies of all other naturally occurring codons that aresynonymous to the respective codon. In some embodiments, each frequencyin a frequency range for a respective codon in the frequency lookuptable is a frequency that the respective codon can be incorporated intoa polynucleotide relative to the total number of amino acids in theencoded polypeptide. In some embodiments, each frequency in a frequencyrange for a respective codon in the frequency lookup table is afrequency that the respective codon can be incorporated into apolynucleotide relative to all other codons in the plurality of codons.

Another method in accordance with this aspect of the invention can beused for designing a test polynucleotide for expression of a polypeptidein an expression system using a frequency table, where the frequencylookup table comprises a plurality of codons. The method comprises (A)constructing a plurality of polynucleotides, each polynucleotide in theplurality of polynucleotides encoding a predetermined polypeptidesequence, where a frequency with which a first codon is used in a firstpolynucleotide in the plurality of polynucleotides, relative to thefrequency with which all other codons in the plurality of codons thatare synonymous to the first codon are used in the first polynucleotide,is different than a frequency with which the first codon is used in asecond polynucleotide in the plurality of polynucleotides, relative tothe frequency with which all other codons in the plurality of codonsthat are synonymous to the first codon are used in the secondpolynucleotide. The method further comprises (B) expressing eachrespective polynucleotide in the plurality of polynucleotidesindividually in the expression system. The method further comprises (C)measuring an expression property value of each respective polynucleotidein the plurality of polynucleotides in the expression system therebyconstructing a dataset, where the dataset comprises, for each respectivepolynucleotide in the plurality of polynucleotides, a representation ofcodon occurrence in the respective polynucleotide and the measuredexpression property value of the respective polynucleotide. The methodfurther comprises (D) computing a model comprising (i) a plurality ofvariables, each variable in the plurality of variables describing afrequency of occurrence of one or more codons in the first plurality ofpolynucleotides, and (ii) a plurality of weights, each weight in theplurality of weights corresponding to one or more variables in theplurality of variables, and where the model describes a variation in themeasured expression property values of the plurality of polynucleotidesfrom the measuring (C) as a function of the plurality of variables andtheir corresponding weights. The method further comprises (E) refiningthe model by principal component analysis in which the plurality ofvariables in the model are reduced to a plurality of latent variables,including a first latent variable, where the plurality of latentvariables collectively account for all or a portion of a variation inthe measured expression property values of the plurality ofpolynucleotides from the measuring C, and where each codon in theplurality of codons has a corresponding load in the first latentvariable. The method further comprises (F) constructing the frequencylookup table for the expression system from the final model, where thelookup table provides a corresponding codon frequency range for each oftwo or more codons in the plurality of codons, and where the codonfrequency range for a codon in the frequency lookup table is determinedby the corresponding load of the codon in the first latent variable. Themethod further comprises (G) defining the test polynucleotide, where thedefining comprises, for each respective codon in the frequency lookuptable, determining whether the respective codon encodes a portion of thepolypeptide sequence. In the method, when the respective codon encodes aportion of the polypeptide, the codon is incorporated into the testpolynucleotide at a frequency of occurrence that is within the frequencyrange specified for the respective codon in the frequency lookup table.The method further comprises (H) outputting the test polynucleotidesequence to a user interface device, a tangible computer readablestorage medium; or displaying the test polynucleotide sequence in userreadable form. In some embodiments, each frequency in a frequency rangefor a respective codon in the frequency lookup table is a frequency thatthe respective codon can be incorporated into the test polynucleotiderelative to all other naturally occurring codons that are synonymous tothe respective codon. In some embodiments, each frequency in a frequencyrange for a respective codon in the frequency lookup table is afrequency that the respective codon can be incorporated into the testpolynucleotide relative to all other codons in the plurality of codons.In some embodiments, each frequency in a frequency range for arespective codon in the frequency lookup table is a frequency that therespective codon can be incorporated into the test polynucleotiderelative to all other naturally occurring codons.

Another aspect provides a method of constructing a frequency lookuptable for an expression system, where the frequency lookup tablecomprises a plurality of sequence elements and optionally a readingframe designation relative to the reading frame of a polypeptide encodedby the polynucleotide. The method comprises constructing a plurality ofpolynucleotides, each polynucleotide in the plurality of polynucleotidesencoding a predetermined polypeptide sequence, where a frequency withwhich a first sequence element is used in a first polynucleotide in theplurality of polynucleotides is different than a frequency with whichthe first sequence element is used in a second polynucleotide in theplurality of polynucleotides. The method further comprises (B)expressing each respective polynucleotide in the plurality ofpolynucleotides individually in the expression system. The methodfurther comprises (C) measuring an expression property value of eachrespective polynucleotide in the plurality of polynucleotides in theexpression system thereby constructing a dataset, where the datasetcomprises, for each respective polynucleotide in the plurality ofpolynucleotides, a representation of sequence element occurrence in therespective polynucleotide and the measured expression property value ofthe respective polynucleotide. The method further comprises (D)computing a model comprising (i) a plurality of variables (e.g., two ormore variables, three or more variables, four or more variables, ten ormore variables, between two and fifty variables, less than fortyvariables, etc.), each variable in the plurality of variables describinga frequency of occurrence of one or more sequence elements in the firstplurality of polynucleotides, and (ii) a plurality of weights (e.g., twoor more weights, three or more weights, four or more weights, ten ormore weights, between two and fifty weights, less than forty weights,etc.), each weight in the plurality of weights corresponding to one ormore variables in the plurality of variables, and where the modeldescribes a variation in the measured expression property values of theplurality of polynucleotides from the measuring (C) as a function of theplurality of variables and their corresponding weights. The methodfurther comprises (E) refining the model by principal component analysisin which the plurality of variables in the model are reduced to aplurality of latent variables, including a first latent variable, wherethe plurality of latent variables collectively account for all or aportion of a variation in the measured expression property values of theplurality of polynucleotides from the measuring C, and where eachsequence element in the plurality of sequence elements has acorresponding load in the first latent variable. The method furthercomprises (F) constructing the frequency lookup table for the expressionsystem from the final model, where the frequency lookup table provides acorresponding sequence element frequency range for each of two or moresequence elements in the plurality of sequence elements, and where thecodon frequency range for a sequence element in the frequency lookuptable is determined by the corresponding load of the sequence element inthe first latent variable. The method further comprises (G) outputtingthe frequency lookup table to a user interface device, a tangiblecomputer readable storage medium; or displaying the frequency lookuptable in user readable form.

Another aspect of the invention provides a method for designing a testpolynucleotide for expression of a polypeptide in an expression system,where the frequency lookup table comprises a plurality of sequenceelements and optionally a reading frame designation relative to thereading frame of a polypeptide encoded by the polynucleotide. In thisaspect, the method comprises (A) constructing a plurality ofpolynucleotides, each polynucleotide in the plurality of polynucleotidesencoding a predetermined polypeptide sequence, where a frequency withwhich a first sequence element is used in a first polynucleotide in theplurality of polynucleotides is different than a frequency with whichthe first sequence element is used in a second polynucleotide in theplurality of polynucleotides. The method further comprises (B)expressing each respective polynucleotide in the plurality ofpolynucleotides individually in the expression system. The methodfurther comprises (C) measuring an expression property value of eachrespective polynucleotide in the plurality of polynucleotides in theexpression system thereby constructing a dataset, where the datasetcomprises, for each respective polynucleotide in the plurality ofpolynucleotides, a representation of sequence element occurrence in therespective polynucleotide and the measured expression property value ofthe respective polynucleotide. The method further comprises (D)computing a model comprising (i) a plurality of variables, each variablein the plurality of variables describing a frequency of occurrence ofone or more sequence elements in the first plurality of polynucleotides,and (ii) a plurality of weights, each weight in the plurality of weightscorresponding to one or more variables in the plurality of variables,and where the model describes a variation in the measured expressionproperty values of the plurality of polynucleotides from the measuring(C) as a function of the plurality of variables and their correspondingweights. The method further comprises (E) refining the model byprincipal component analysis in which the plurality of variables in themodel are reduced to a plurality of latent variables, including a firstlatent variable, where the plurality of latent variables collectivelyaccount for all or a portion of a variation in the measured expressionproperty values of the plurality of polynucleotides from the measuring(C), and where each sequence element in the plurality of sequenceelements has a corresponding load in the first latent variable. Themethod further comprises (F) constructing the frequency lookup table forthe expression system from the final model, where the frequency lookuptable provides a corresponding sequence element frequency range for eachof two or more sequence elements in the plurality of sequence elements,and where the sequence element frequency range for a sequence element inthe frequency lookup table is determined by the corresponding load ofthe sequence element in the first latent variable. The method furthercomprises (G) defining the test polynucleotide, where the definingcomprises, for each respective sequence element in the frequency lookuptable, determining whether the respective sequence element encodes aportion of the polypeptide sequence, where, when the respective sequenceelement encodes a portion of the polypeptide, the sequence element isincorporated into the test polynucleotide at a frequency of occurrencethat is within the frequency range specified for the respective sequenceelement in the frequency lookup table. The method further comprises (H)outputting the test polynucleotide sequence to a user interface device,a tangible computer readable storage medium; or displaying the testpolynucleotide sequence in user readable form.

5.6 DESIGN OF NEW SEQUENCE ELEMENT FREQUENCY LOOKUP TABLES

Analyses of the expression properties and frequencies of codons or othersequence elements in a codon variant set of polynucleotides can be usedto identify frequencies or ranges of frequencies for sequence elements,such as codons, codon pairs and nucleotide triplets in the +3 readingframe within a polynucleotide that result in superior expressionproperties. The results of this analysis can be formulated as afrequency lookup table to be used in the design of polynucleotidesequences. In this way the expression properties of one codon variantset may be translated to design principles that can be used in thedesign of polynucleotides encoding polypeptides that are unrelated tothe initial codon variant set. The extraction of polynucleotide designprinciples from codon variant sets are an aspect of the invention.

5.6.1 Sequence Element Frequency Lookup Tables Based Directly onVariants in a Codon Variant Set

In a preferred embodiment of the invention, the absolute or relativefrequencies of one or more sequence elements (e.g., codons) in thevariant in the codon valiant set that has the most desirable expressionproperty or properties are used as sequence element target frequenciesfor the design of other polynucleotides. The sequence element targetfrequencies can be encoded as a sequence element frequency lookup table,or as a codon frequency matrix, or otherwise used to calculate a desiredscore for a polynucleotide encoding a polypeptide.

Such sequence element target frequencies can be used to designpolynucleotides to encode polypeptides that may or may not be related tothe polypeptides present in the codon variant set. The frequency of eachsequence element within the designed polynucleotides will preferably bebetween 1.5 times and 0.5 times the target codon frequencies, morepreferably between 1.25 times and 0.75 times the target codonfrequencies. In some embodiments the acceptable range of frequencies ofsequence elements is explicitly defined.

In a variation of this embodiment of the invention, target codonfrequencies can be derived from the 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9or 10 variants in the codon-variant set that have the most desirableexpression property or properties, for example by calculating anarithmetic or geometric mean value for the codon frequency found inthese best variants.

5.6.2 Adjusting Frequency Lookup Tables

In some instances, sequence element target frequencies for the design ofother polynucleotides can be calculated by more complexsequence-expression modeling. For example relative or absolutefrequencies for one or more of the codons for amino acids with multiplecodon choices, or other sequence elements such as nucleotide triplets inthe +3 reading frame (which encodes the “wobble” or third base of onecodon and the first two bases of the following codon) and nucleotidehexamers in the +1 reading frame (which encode an adjacent pair ofcodons or “codon pair”) can be used as the input independent variablesfor sequence-expression modeling. In this embodiment the resultingindependent variable matrix contains one or more discrete values foreach of the variables. Multivariate regression analysis or othersequence-expression analysis can then be applied to determinecorrelation of an expression property with the relative or absolutefrequencies for one or more of the codons for amino acids with multiplecodon choices, or other sequence elements. The resulting model can showwhich codon frequencies for each amino acid correlate most strongly withdesired expression properties, or which other sequence elementscorrelate most strongly with desired expression properties. A new codonfrequency lookup table can be constructed, for example, by using thecodon frequency for each amino acid that the sequence-expression modelcorrelates most strongly with desired expression properties.

In another embodiment of the invention the degree of bias relative to areference relative frequency of synonymous codons can be used as theinput independent variables for sequence-expression modeling. A codonvariant set can be created by varying the codon bias for each amino acidsystematically using Design of Experiments methodology. In thisembodiment the resulting independent variable matrix would contain twoor more discrete values for each of the 18 variables (e.g. “more” or“less” biased). Multivariate regression analysis could then be appliedto determine correlation of the biases by amino acid with expression.The resulting model would suggest which amino acid codon sets should bemore and which should be less biased. An optimal combination of thebiases that show the strongest positive contribution to expression canthen be constructed and used as an optimized codon frequency lookuptable.

Codon biases calculated from analyzing sequence-expression data can inturn be used as the starting point for another experiment to determinewhether more or less bias for each amino acid relative to this new tableis beneficial. Iterations of this procedure could be used to find anoptimum for each amino acid and, thus, an optimal complete codon table.

Alternatively the new frequency lookup tables or biases or matrices cansimply be used to determine sequence element target frequencies in newpolynucleotides.

5.6.3 Frequency Lookup Tables with Frequency Ranges

There are frequently multiple design criteria that must be followed inthe design of synthetic polynucleotides. For polynucleotides that encodepolypeptides, one constraint is that the polynucleotide should express.Other constraints can include (i) exclusion of a restriction sitesequence; (ii) incorporation of a restriction site sequence in thepolynucleotide sequence; (iii) a designation of a target G+C content inthe polynucleotide sequence; (iv) an allowable length of a sub-sequencethat is exactly repeated within either strand of the polynucleotidesequence; (v) an allowable annealing temperature of any sub-sequence toany other sub-sequence within either strand of the polynucleotidesequence; (vi) exclusion of a hairpin turn in the polynucleotidesequence; (vii) exclusion of a repeat element in the polynucleotidesequence; (viii) exclusion of a ribosome binding site in thepolynucleotide sequence; (ix) exclusion of a polyadenylation signal inthe polynucleotide sequence; (x) exclusion of a splice site in thepolynucleotide sequence; (xi) exclusion of an open reading frame greaterthan a certain length in each possible 5′ to 3′ reading frame in thepolynucleotide sequence; (xii) exclusion of a polynucleotide sequencethat facilitates RNA degradation in the polynucleotide sequence; (xiii)exclusion of an RNA polymerase termination signal in the polynucleotidesequence; (xiv) exclusion of a transcriptional promoter in thepolynucleotide sequence; (xv) exclusion of an immunostimulatory sequencein the polynucleotide sequence; (xvi) incorporation of animmunostimulatory sequence in the polynucleotide sequence; (xvii)exclusion of an RNA methylation signal in the polynucleotide sequence;(xviii) exclusion of a selenocysteine incorporation signal in thepolynucleotide sequence; (xix) exclusion of an RNA editing sequence inthe polynucleotide sequence; (xx) exclusion of an RNAi-targeted sequencein the polynucleotide sequence; (xxi) exclusion of an inverted repeatwithin the polynucleotide sequence and/or (xxii) exclusion of a DNAmethylation signal in the polynucleotide sequence

A polynucleotide sequence can be designed to fit many design criteriasimultaneously by defining quantifiable polynucleotide sequenceproperties or combination of properties. The properties can also bedescribed as the presence or absence of a feature (represented as abinary or Boolean term). For example the design criteria can be achievedby a Monte Carlo algorithm: (1) select an initial codon sequence toencode a polypeptide; (2) quantify the one or more polynucleotidesequence properties of interest; (3) identify a possible change to thecodon sequence that does not change the encoded polypeptide; (4)quantify the one or more polynucleotide sequence properties of interestfor the polynucleotide sequence after the change in codon sequence; (5)accept or reject the change based on a function of the difference invalues for the one or more polynucleotide properties of interest for theoriginal and the unchanged codon sequences; and (6) repeat this processuntil a desired value for the one or more polynucleotide sequenceproperties of interest is obtained. Searches for codon sequences thathave defined values for one or more quantifiable polynucleotide sequenceproperty can also be performed by evolving the sequence using geneticalgorithms or genetic algorithms in combination with monte carloalgorithms, or other stochastic searches such as simulated annealing,Boltzmann learning.

One example of how to perform such a design is as follows:

Select an initial polynucleotide sequence encoding a polypeptide; then

for 10000 iterations

{

-   -   i. Calculate the values for all of the quantifiable        polynucleotide sequence properties corresponding to design        criteria;    -   ii. Compare these values with the desired values for all of the        quantifiable polynucleotide sequence properties corresponding to        design criteria;    -   iii. Randomly choose one codon in the polynucleotide;    -   iv. Randomly change the codon for a synonymous codon;    -   v. For the new polynucleotide calculate the values for all of        the quantifiable polynucleotide sequence properties        corresponding to design criteria;    -   vi. Compare these values with the desired values for all of the        quantifiable polynucleotide sequence properties corresponding to        design criteria;    -   vii. If the values for the polynucleotide created in step iv)        more closely match the design criteria than the polynucleotide        of step i), accept the change from step iv), else, dismiss the        changes and retain original values.

}

Output a polynucleotide sequence.

In some embodiments, it is advantageous to precisely control thefrequencies of some sequence elements while less precisely controllingthe frequencies of other sequence elements. In particular it isadvantageous to less precisely control the frequencies of sequenceelements whose frequencies are less critical for determining theexpression property of the polynucleotide. If there are multiple designcriteria in addition to sequence element frequency, relaxing the designconstraints on sequence elements whose frequencies are less functionallycritical increases the sequence possibilities that can be used to meetthe other design criteria.

Sequence-expression modeling can produce values for the absolute orrelative frequency for each codon that best correlates with desiredexpression properties. It can also produce a measure of the importanceof each sequence element in determining the expression properties, orthe acceptable range of frequencies for each sequence element that areconsistent with certain desired expression properties. Frequency lookuptables can be created from sequence-expression relationships to describea target range of relative or absolute frequencies for each sequenceelement instead of the single value for each sequence element that isfound in codon bias tables that describe the frequencies of codons foundin naturally occurring sequences or genomes. These frequency lookuptables can thus provide a target range for the frequency of eachsequence element in a designed polynucleotide, rather than a singletarget value. This is particularly advantageous when the frequencies ofsome sequence elements have strong correlations with desired expressionproperties and the frequencies of other sequence elements appear to beless important.

5.6.4 Use of Frequency Lookup Tables to Design a Final Polynucleotide

Another aspect provides methods of using one or more frequency lookuptables to design a final polynucleotide that encodes an amino acidsequence of a predetermined polypeptide. For instance, in one suchmethod a frequency lookup table corresponding to an expression system isobtained. The frequency lookup table comprises a plurality of sequenceelements and a plurality of frequency ranges. Each frequency range inthe plurality of frequency ranges is for a corresponding sequenceelement in the plurality of sequence elements. Each respective frequencyrange in the plurality of frequency ranges specifies a range offrequencies that the sequence element corresponding to the respectivefrequency range can occur in a polynucleotide that is to be expressed inthe expression system. Each sequence element in the plurality ofsequence elements is optionally associated with a frame designation inthe frequency lookup table. For instance, the frequency lookup table mayspecify a frequency range for a given sequence element in the readingframe that encodes the amino acid sequence of a predeterminedpolypeptide. In this instance, the frequency lookup table is onlyconstraining the frequency range of the sequence element in the readingframe that encodes the amino acid sequence of the predeterminedpolypeptide, not other frames. The frequency lookup table does notconstrain the frequency that the sequence element appears in otherframes. For example, consider the case where the sequence element is thecodon TTT and the frequency lookup table specifies that the frame forTTT is the reading frame that encodes the amino acid sequence of thepredetermined polypeptide. The sequence TTT may appear in other readingframes (e.g., split across two codons) but the frequency lookup tabledoes not place a constraint on such occurrences. Of course, thefrequency lookup table can be used to place frequency ranges on multipleframes for a given sequence element. Further, the frequency lookup tablecan be used to place a first frequency range on a given sequence elementwhen the sequence element is in one frame and a second frequency rangeon a given sequence element when the sequence element is in a secondframe, where the two frequency ranges are different. By different, whatis meant here is that the two frequency ranges have a different maximumand/or minimum. Frequency ranges can be given as absolute numbers (e.g.,a maximum or minimum specific number of occurrences allowed in thedesignated frame), relative frequencies (e.g., in the case where thesequence element is a codon, the minimum and maximum percentage ofoccurrence of the codon relative to all other synonymous codonsregardless of the number of times the corresponding amino acid appearsin the polynucleotide sequence), absolute frequencies (e.g., the minimumand maximum percentage of occurrence of the sequence element relative toall other sequence elements in the frequency lookup table), absoluterelative frequencies (e.g., the minimum and maximum percentage ofoccurrence of the sequence element relative to a list of other sequenceelements specified in the frequency lookup table), or some other metricthat involves a range of allowed occurrences of the sequence element. Anexample of the case of absolute relative frequency, the frequency lookuptable specifies a first sequence element, an optional frame, and a listof sequence elements that serve as a basis for constraining theoccurrence of the first sequence element in the polynucleotide. Forexample, consider the case where the sequence element is labeled “A” andthe list of sequence elements consists of the sequence elements labeled“B,” “C,” and “D.” In this case, the frequency range allowed or “A” isrelative to the sum total of the occurrence of “B,” “C,” and “D” in thedesignated reading frame of the polynucleotide.

In the method a test polynucleotide that encodes the amino acid sequenceis defined. Further, a score for the test polynucleotide is determinedusing the frequency lookup table, where the score determined for thetest polynucleotide is collectively determined by a plurality ofagreement scores, each agreement score in the plurality of agreementscores being a measure of agreement between (i) an actual sequenceelement frequency of a respective sequence element in the plurality ofsequence elements in the test polynucleotide and (ii) a frequency rangespecified for the respective sequence element in the frequency lookuptable or a measure of agreement between (iii) an actual quantifiablesequence property of the test polynucleotide and (iv) a value rangespecified for the respective quantifiable sequence property in thelookup table. For example, consider a lookup table that contains threesequence elements and three corresponding frequency ranges. A testpolynucleotide in this example is scored by determining three agreementscores. Each agreement score is a function of how well the frequency ofoccurrence in the test polynucleotide of one of the three sequenceelements fits into the frequency ranges specified for the sequenceelement in the frequency lookup table. In some embodiments, theagreement score approaches a predetermined ideal score when thefrequency of occurrence of the sequence element in the testpolynucleotide approaches the middle of the frequency range specified inthe frequency lookup table for the sequence element. In someembodiments, the agreement score approaches a predetermined ideal scorewhen the frequency of occurrence of the sequence element in the testpolynucleotide approaches the minimum allowed frequency in the frequencyrange specified in the frequency lookup table for the sequence element.In some embodiments, the agreement score approaches a predeterminedideal score when the frequency of occurrence of the sequence element inthe test polynucleotide approaches the maximum allowed frequency in thefrequency range specified in the frequency lookup table for the sequenceelement. By “approaches a predetermined ideal score” it is meant thatthe score becomes closer to a score that is considered to be optimal.The optimal score can, for example, be a high value, such as 100, or alow value such as zero. Thus, consider the case where the optimal scoreis zero and the agreement score approaches a predetermined ideal scorewhen the frequency of occurrence of the sequence element in the testpolynucleotide approaches the maximum allowed frequency in the frequencyrange specified in the frequency lookup table for the sequence element.In this case, the agreement score approaches zero when the frequency ofoccurrence of the sequence element in the test polynucleotide approachesthe maximum allowed frequency in the frequency range specified in thefrequency lookup table for the sequence element.

In some embodiments, each of the agreement scores (from each of thesequence elements in the frequency lookup table) are summed together toobtain the score for the test polynucleotide. In some embodiments, eachof the agreement scores (from each of the sequence elements in thefrequency lookup table) are summed together to obtain the score for thetest polynucleotide but individual agreement scores are weighted by howoften they appear in the test polynucleotide in the reading framespecified by the frequency lookup table. In some embodiments, anymathematical function is used to combine the agreement scores into thescore for the test polynucleotide. For example, the agreement scores canbe multiplied together. In some embodiments the sum of the squares ofthe differences between all of the (i) actual sequence elementfrequencies of a respective sequence element in the plurality ofsequence elements in the test polynucleotide and (ii) the frequencyrange specified for the respective sequence element in the frequencylookup table and/or the differences between all of the (iii) actualquantifiable sequence property of the test polynucleotide and (iv) avalue range specified for the respective quantifiable sequence propertyin the lookup table. In some embodiments, the predetermined ideal scorefor the test polynucleotide is a low value and the lower the scorecomputed for the test polynucleotide (by combination of all theindividual agreement scores), the closer this score is to thepredetermined ideal score. In some embodiments, the predetermined idealscore for the test polynucleotide is a high value and the higher thescore computed for the test polynucleotide (by combination of all theindividual agreement scores), the closer this score is to thepredetermined ideal score.

In the method, the test polynucleotide is then processed. Suchprocessing assumes a case where there are multiple instances of thedefining and determining described above, with each instance of thedefining and determining resulting in a different test polynucleotidewith a score. The processing is used to decide which test polynucleotideto keep and which test polynucleotides to reject based on theirrespective scores. In some embodiments, an instance of this processingis run after each instance of the defining and determining in order todecide whether to keep the newly determined test polynucleotide or toreject the newly determined test polynucleotide in favor of a previouslydetermined polynucleotide. In some embodiments, this processingcomprises accepting the recently determined test polynucleotide when (i)the test polynucleotide has a score that is closer to a predeterminedideal score than any other score assigned to a test polynucleotide. Ifthere is no other test polynucleotide that has been determined, then thetest polynucleotide is deemed to have a score that is closer to apredetermined ideal score than any other score assigned to a testpolynucleotide. The test polynucleotide is rejected when another testpolynucleotide in an instance of the determining (e.g., computed beforethe last instance of the determining) has a score that is closer to anideal score than the score assigned to the test polynucleotide in thelast instance of defining and determining. In some embodiments, theideal score is a low value (e.g. zero or some negative value). In someembodiments, the ideal score is a hight value (e.g. one or some otherpositive value).

The defining, determining, and processing are repeated until an exitcondition is achieved, where the test polynucleotide to be accepted bythe last instance of the processing before the exit condition isachieved is deemed to be the final polynucleotide. The finalpolynucleotide is then outputted to a user interface device, a tangiblecomputer readable storage medium, or the final polynucleotide isdisplayed in user readable form. In some embodiments, the exit conditionis the repetition of the defining, determining, and processing apredetermined number of times (e.g., two or more times, three or moretimes, four or more times, between five and one hundred times, more thanten times, or than one thousand times, more than one hundred thousandtimes, or more than one million times).

In some embodiments, the exit condition is the repetition of thedefining, determining, and processing until a score for a testpolynucleotide computed in an instance of the determining is within apredetermined threshold of an ideal score. The actual value of thepredetermined threshold is application dependent. For instance, thepredetermined threshold will depend upon the mathematical function thatis used to combine individual agreement scores into the score for a testpolynucleotide.

An advantage of the methods described in this section is that thedefining of a test polynucleotide can accommodate many different designcriteria. These design criteria can be absolute, such as the exclusionof a specific sequence element or they can be constrained to frequencyranges specified in a frequency lookup table, or they can be constrainedto specific values specified in a quantifiable sequence property lookuptable, or they can be associated with specific values for scores orpenalties. In some embodiments, the designing of a test polynucleotideabove further comprises (i) exclusion of a restriction site sequence inthe test polynucleotide, (ii) incorporation of a restriction sitesequence in the test polynucleotide, (iii) a designation of a target G+Ccontent in the test polynucleotide, (iv) exclusion of a sub-sequence,longer than an allowable length, that is exactly repeated within eitherstrand of the test polynucleotide, (v) exclusion of a first sub-sequencethat can anneal at a greater than an allowable annealing temperature toa second sub-sequence within either strand of the test polynucleotide,(vi) exclusion of a hairpin turn in the test polynucleotide, (vii)exclusion of a repeat element in the test polynucleotide, (viii)exclusion of a ribosome binding site in the test polynucleotide, (ix)exclusion of a polyadenylation signal in the test polynucleotide, (x)exclusion of a splice site in the test polynucleotide, (xi) exclusion ofan open reading frame in each possible reading frame in the testpolynucleotide other than a reading frame encoding the amino acidsequence, (xii) exclusion of a polynucleotide sequence that facilitatesRNA degradation in the test polynucleotide, (xiii) exclusion of an RNApolymerase termination signal in the test polynucleotide, (xiv)exclusion of a transcriptional promoter in the test polynucleotide, (xv)exclusion of an immunostimulatory sequence in the test polynucleotide,(xvi) incorporation of an immunostimulatory sequence in the testpolynucleotide, (xvii) exclusion of an RNA methylation signal in thetest polynucleotide, (xviii) exclusion of a selenocysteine incorporationsignal in the test polynucleotide, (xix) exclusion of an RNA editingsequence in the test polynucleotide, (xx) exclusion of an RNAi-targetedsequence in the test polynucleotide, (xxi) exclusion of an invertedrepeat within the first x number of nucleotides encoding the amino acidsequence in the test polynucleotide, where x is any value between onenucleotide and one hundred nucleotides, or (xxii) exclusion of a DNAmethylation signal in the test polynucleotide.

In some embodiments, the score determined for a test polynucleotide inan instance of the determining is further determined by (i) exclusion ofa restriction site sequence in the test polynucleotide, (ii)incorporation of a restriction site sequence in the test polynucleotide,(iii) a designation of a target G+C content in the test polynucleotide,(iv) exclusion of a sub-sequence, longer than an allowable length, thatis exactly repeated within either strand of the test polynucleotide, (v)exclusion of a first sub-sequence that can anneal at a greater than anallowable annealing temperature to a second sub-sequence within eitherstrand of the test polynucleotide, (vi) exclusion of a hairpin turn inthe test polynucleotide, (vii) exclusion of a repeat element in the testpolynucleotide, (viii) exclusion of a ribosome binding site in the testpolynucleotide, (ix) exclusion of a polyadenylation signal in the testpolynucleotide, (x) exclusion of a splice site in the testpolynucleotide, (xi) exclusion of an open reading frame in each possiblereading frame in the test polynucleotide other than a reading frameencoding the amino acid sequence, (xii) exclusion of a polynucleotidesequence that facilitates RNA degradation in the test polynucleotide,(xiii) exclusion of an RNA polymerase termination signal in the testpolynucleotide, (xiv) exclusion of a transcriptional promoter in thetest polynucleotide, (xv) exclusion of an immunostimulatory sequence inthe test polynucleotide, (xvi) incorporation of an immunostimulatorysequence in the test polynucleotide, (xvii) exclusion of an RNAmethylation signal in the test polynucleotide, (xviii) exclusion of aselenocysteine incorporation signal in the test polynucleotide, (xix)exclusion of an RNA editing sequence in the test polynucleotide, (xx)exclusion of an RNAi-targeted sequence in the test polynucleotide, (xxi)exclusion of an inverted repeat within the first x nucleotides encodingthe amino acid sequence in the test polynucleotide, where x is anynumber between one nucleotide and one hundred nucleotides, or (xxii)exclusion of a DNA methylation signal in the test polynucleotide. Forexample, in some embodiments, the score for a test polynucleotide isadjusted so that it is closer to an ideal value when the testpolynucleotide does not have a restriction sequence that is to beexcluded from test polynucleotide. In another example, in someembodiments, the score for a test polynucleotide is adjusted so that itis closer to an ideal value when the test polynucleotide incorporates arestriction sequence that is to be included in the test polynucleotide.In another example, in some embodiments, the score for a testpolynucleotide is adjusted so that it is further away from an idealvalue when the test polynucleotide contains a sub-sequence, longer thanan allowable length, that can be exactly repeated within either strandof the test polynucleotide. Such a sub-sequence may be undersirablebecause the repeats of the sub-sequence may anneal to each other andprevent translation of the polynucleotide.

In some embodiments, the frequency lookup table further comprises amodel that computes an estimated expression property value for anexpression property of the predetermined polypeptide as a function of asequence of a test polynucleotide for the expression system, and wherethe score determined for a respective test polynucleotide in an instanceof the determining is further determined by the estimated expressionproperty value for the predetermined polypeptide calculated by the modelbased on a nucleotide sequence of the respective test polynucleotide. Insome embodiments, the expression property is an estimate of a totalamount of protein encoded by the respective test polynucleotide whenexpressed in the expression system in a predetermined period of time. Insome embodiments, the expression property is an estimate of a totalamount of active protein encoded by the respective test polynucleotidewhen expressed in the expression system in a predetermined period oftime. In some embodiments, the expression property is an estimate of atotal amount of soluble protein encoded by the respective testpolynucleotide when expressed in the expression system in apredetermined period of time. This model can be created by evaluation ofa codon variant set (plurality of nucleotides) in the expression systembeing used to express the final polynucleotide using any of the methodsdisclosed herein.

In some embodiments, the sequence element in the plurality of sequenceelements consists of a codon. In some embodiments, a sequence element inthe plurality of sequence elements consists of a codon pair. A codonpair is a predetermined first codon and predetermined second codon thatare adjacent to each other and are in the same reading frame. In someembodiments, a sequence element in the plurality of sequence elementsconsists of a nucleotide triplet in the +3 reading frame (which encodesthe “wobble” or third base of one codon and the first two bases of thefollowing codon)

In some embodiments, the plurality of sequence elements consists of upto five, up to ten, up to fifteen, up to twenty, up to twenty-five, orup to thirty naturally occurring codons and the frequency lookup tableplaces no restrictions on the frequency of occurrence of a codon in atest sequence when the codon is not in the frequency lookup table.

Another aspect provides a computer-readable medium storing one or morecomputer programs executable by a computer, the one or more computerprograms collectively comprising instructions for performing any of themethods described in this section. Another aspect provides an apparatuscomprising one or more processors and a memory, coupled to the one ormore processors, the memory storing one or more computer programs thatindividually or collectively comprise instructions for performing any ofthe methods disclosed in this section.

5.6.5 Use of Frequency Lookup Tables and a Genetic Algorithm to Design aFinal Polynucleotide

Another aspect of the invention comprises designing a finalpolynucleotide that encodes an amino acid sequence of a predeterminedpolynucleotide using a genetic algorithm. In the method, a frequencylookup table corresponding to an expression system is obtained. Thefrequency lookup table comprises a plurality of sequence elements and aplurality of frequency ranges. Each frequency range in the plurality offrequency ranges is for a corresponding sequence element in theplurality of sequence elements. Each respective frequency range in theplurality of frequency ranges specifies a range of frequencies that thesequence element corresponding to the respective frequency range canoccur in a polynucleotide that is to be expressed in the expressionsystem. Each sequence element in the plurality of sequence elements isoptionally associated with a frame designation in the frequency lookuptable. In some embodiments, the plurality of sequence elements is fiveor more sequence elements, ten or more sequence elements, fifteen ormore sequence elements, twenty or more sequence elements, twenty-five ormore sequence elements, between five and ten sequence elements, or lessthan fifty sequence elements. In the methods, a set of testpolynucleotides is defined, where each test polynucleotide in the set oftest polynucleotides encodes the amino acid sequence. Then, a fit ofeach test polynucleotide in the set of test polynucleotides isdetermined, where the fit of a respective test polynucleotide in the setof test polynucleotides is determined by a combination of a plurality ofagreement scores, each agreement score in the plurality of agreementscores being a measure of agreement between (i) an actual sequenceelement frequency of a respective sequence element in the plurality ofsequence elements in the respective test polynucleotide sequence and(ii) a frequency range specified for the respective sequence element inthe frequency lookup table or a measure of agreement between (iii) anactual quantifiable sequence property of the test polynucleotide and(iv) a value range specified for the respective quantifiable sequenceproperty in the lookup table. Then a determination as to whether an exitcondition has been satisfied is made. When the exit condition has notbeen satisfied, the set of test polynucleotides is enriched by removinga portion of the polynucleotides from the set of test polynucleotidesbased on a respective fit determined for each test polynucleotide in theset of test polynucleotides. When the exit condition has not beensatisfied, the set of test polynucleotides is also enriched by adding asubset of test polynucleotides to the set of test polynucleotides, whereeach test polynucleotide in the subset of test polynucleotides isderived from one or more test polynucleotides already in the set of testpolynucleotides and where each test polynucleotide in the subset of testpolynucleotides encodes the amino acid sequence of the predeterminedpolypeptide. The steps of determining a fit, determining an exitcondition, enriching by removing polynucleotides, and enriching byadding polynucleotides is repeated until the exit condition has beensatisfied. A final polynucleotide from the set of test polynucleotidesis outputted to a user interface device, a tangible computer readablestorage medium; or the final polynucleotide is displayed in userreadable form. In some embodiments, the final polynucleotide is a testpolynucleotide in the set of test polynucleotides that has a fit that iscloser to an ideal fit than any other test polynucleotide in the set oftest polynucleotides. In some embodiments the exit condition is therepetition of the determining a fit, the determining an exit condition,the enriching by removal, and the enriching by repeating a predeterminednumber of times. In some embodiments, the exit condition is therepetition of the determining a fit, the determining whether an exitcondition has not been satisfied, the enriching through removal ofpolynucleotides, and the enriching through adding derivedpolynucleotides until a fit of a test polynucleotide computed in aninstance of the determining whether an exit condition is within apredetermined threshold of an ideal score. The exact value of thepredetermined threshold is application dependent. Further, in someembodiments the ideal score is a minimum possible score, a maximumpossible score, or a specific predetermined value.

In some embodiments, the defining a set of test polynucleotidescomprises exclusion of a restriction site sequence in a testpolynucleotide in the set of test polynucleotides, (ii) incorporation ofa restriction site sequence in a test polynucleotide in the set of testpolynucleotides, (iii) a designation of a target G+C content in a testpolynucleotide in the set of test polynucleotides, (iv) exclusion of asub-sequence, longer than an allowable length, that can be exactlyrepeated within either strand of a test polynucleotide in the set oftest polynucleotides, (v) exclusion of a first sub-sequence that cananneal at a greater than an allowable annealing temperature to a secondsub-sequence within either strand of a test polynucleotide in the set oftest polynucleotides, (vi) exclusion of a hairpin turn in a testpolynucleotide in the set of test polynucleotides, (vii) exclusion of arepeat element in a test polynucleotide in the set of testpolynucleotides, (viii) exclusion of a ribosome binding site in a testpolynucleotide in the set of test polynucleotides, (ix) exclusion of apolyadenylation signal in a test polynucleotide in the set of testpolynucleotides, (x) exclusion of a splice site in a test polynucleotidein the set of test polynucleotides, (xi) exclusion of an open readingframe in each possible reading frame in a test polynucleotide in the setof test polynucleotides other than the reading frame encoding thepredetermined polypeptide, (xii) exclusion of a polynucleotide sequencethat facilitates RNA degradation in a test polynucleotide in the set oftest polynucleotides, (xiii) exclusion of an RNA polymerase terminationsignal in a test polynucleotide in the set of test polynucleotides,(xiv) exclusion of a transcriptional promoter in a test polynucleotidein the set of test polynucleotides, (xv) exclusion of animmunostimulatory sequence in a test polynucleotide in the set of testpolynucleotides, (xvi) incorporation of an immunostimulatory sequence ina test polynucleotide in the set of test polynucleotides, (xvii)exclusion of an RNA methylation signal in a test polynucleotide in theset of test polynucleotides, (xviii) exclusion of a selenocysteineincorporation signal in a test polynucleotide in the set of testpolynucleotides, (xix) exclusion of an RNA editing sequence in a testpolynucleotide in the set of test polynucleotides, (xx) exclusion of anRNAi-targeted sequence in a test polynucleotide in the set of testpolynucleotides, (xxi) exclusion of an inverted repeat within the first45 nucleotides encoding the amino acid sequence in a test polynucleotidein the set of test polynucleotides or (xxii) exclusion of a DNAmethylation signal in a test polynucleotide in the set of testpolynucleotides.

In some embodiments, the fit that is determined for a testpolynucleotide in an instance of the determining whether an exitcondition has been satisfied is further determined by (i) exclusion of arestriction site sequence in a test polynucleotide in the testpolynucleotide, (ii) incorporation of a restriction site sequence in thetest polynucleotide, (iii) a designation of a target G+C content in thetest polynucleotide, (iv) exclusion of a sub-sequence, longer than anallowable length, that can be exactly repeated within either strand ofthe test polynucleotide, (v) exclusion of a first sub-sequence that cananneal at a greater than an allowable annealing temperature to a secondsub-sequence within either strand of the test polynucleotide, (vi)exclusion of a hairpin turn in the test polynucleotide, (vii) exclusionof a repeat element in the test polynucleotide, (viii) exclusion of aribosome binding site in the test polynucleotide, (ix) exclusion of apolyadenylation signal in the test polynucleotide, (x) exclusion of asplice site in the test polynucleotide, (xi) exclusion of an openreading frame in each possible reading frame in the test polynucleotidein other than the reading frame encoding the predetermined polypeptide,(xii) exclusion of a polynucleotide sequence that facilitates RNAdegradation in the test polynucleotide, (xiii) exclusion of an RNApolymerase termination signal in the test polynucleotide, (xiv)exclusion of a transcriptional promoter in the test polynucleotide, (xv)exclusion of an immunostimulatory sequence in the test polynucleotide,(xvi) incorporation of an immunostimulatory sequence in the testpolynucleotide, (xvii) exclusion of an RNA methylation signal in thetest polynucleotide, (xviii) exclusion of a selenocysteine incorporationsignal in the test polynucleotide, (xix) exclusion of an RNA editingsequence in the test polynucleotide, (xx) exclusion of an RNAi-targetedsequence in the test polynucleotide, (xxi) exclusion of an invertedrepeat within the first x nucleotides encoding the amino acid sequencein the test polynucleotide, where x is any number between one nucleotideand one hundred nucleotides, or (xxii) exclusion of a DNA methylationsignal in the test polynucleotide. For example, in some embodiments, thefit for a test polynucleotide is adjusted so that it is closer to anideal value when the test polynucleotide does not have a restrictionsequence that is to be excluded from test polynucleotide. In anotherexample, in some embodiments, the fit for a test polynucleotide isadjusted so that it is closer to an ideal value when the testpolynucleotide incorporates a restriction sequence that is to beincluded in the test polynucleotide. In another example, in someembodiments, the fit for a test polynucleotide is adjusted so that it isfurther away from an ideal value when the test polynucleotide contains asub-sequence, longer than an allowable length, that can be exactlyrepeated within either strand of the test polynucleotide. Such asub-sequence may be undersirable because the repeats of the sub-sequencemay anneal to each other and prevent translation of the polynucleotide.

In some embodiments, the frequency lookup table further comprises amodel that computes an estimated expression property value for anexpression property of the predetermined polypeptide as a function of anucleotide sequence of a test polynucleotide, and wherein the fitdetermined for a respective test polynucleotide in an instance of thedetermining a fit of a respective test polynucleotide in the set of testpolynucleotides is further determined by the estimated expressionproperty value for the predetermined polypeptide calculated by the modelbased on a nucleotide sequence of the respective test polynucleotide. Insome embodiments, the expression property is an estimate of a totalamount of protein encoded by the respective test polynucleotide whenexpressed in the expression system in a predetermined period of time. Insome embodiments, the expression property is an estimate of a totalamount of active protein encoded by the respective test polynucleotidewhen expressed in the expression system in a predetermined period oftime. In some embodiments, the expression property is an estimate of atotal amount of soluble protein encoded by the respective testpolynucleotide when expressed in the expression system.

In some embodiments, the sequence element in the plurality of sequenceelements consists of a codon. In some embodiments, a sequence element inthe plurality of sequence elements consists of a codon pair. A codonpair is a predetermined first codon and predetermined second codon thatare adjacent to each other and are in the same reading frame. In someembodiments, a sequence element in the one or more sequence elements isa nucleotide triplet in the +3 reading frame (encoding the “wobble” orthird base of one codon and the first two bases of the following codon).

In some embodiments, the plurality of sequence elements consists of upto five, up to ten, up to fifteen, up to twenty, up to twenty-five, orup to thirty naturally occurring codons and the frequency lookup tableplaces no restrictions on the frequency of occurrence of a codon in atest sequence when the codon is not in the frequency lookup table.

Another aspect provides a computer-readable medium storing one or morecomputer programs executable by a computer, the one or more computerprograms collectively comprising instructions for performing any of themethods described in this section. Another aspect provides an apparatuscomprising one or more processors and a memory, coupled to the one ormore processors, the memory storing one or more computer programs thatindividually or collectively comprise instructions for performing any ofthe methods disclosed in this section.

5.6.6 Use of Frequency Lookup Tables and a Global Optimization Algorithmto Design a Final Polynucleotide

Another aspect of the invention provides methods for designing a finalpolynucleotide that encodes an amino acid sequence of a predeterminedpolypeptide. In some embodiments, a frequency lookup table correspondingto an expression system is obtained. The frequency lookup tablecomprises a plurality of sequence elements and a plurality of frequencyranges. Each frequency range in the plurality of frequency ranges is fora corresponding sequence element in the plurality of sequence elements.Each respective frequency range in the plurality of frequency rangesspecifies a range of frequencies that the sequence element correspondingto the respective frequency range can occur in a polynucleotide that isto be expressed in the expression system. Each sequence element in theplurality of sequence elements is optionally associated with a framedesignation in the frequency lookup table.

In some embodiments, a multivariate space is searched using a globaloptimization algorithm. In some embodiments, the plurality of sequenceelements is five or more sequence elements, ten or more sequenceelements, fifteen or more sequence elements, twenty or more sequenceelements, twenty-five or more sequence elements, between five and tensequence elements, or less than fifty sequence elements.

The multivariate space comprising a plurality of variables, eachvariable in the plurality of variables for quantifying a fit between (i)a frequency of occurrence in a polynucleotide of a respective sequenceelement in the plurality of sequence elements and (ii) the frequencyrange for the respective sequence element in the frequency lookup table,where the global optimization algorithm assigns a score to eachrespective test polynucleotide in a plurality of test polynucleotides inorder to find the final model, each respective score being aquantification of the fit of the respective test polynucleotide, andeach test polynucleotide in the plurality of test polynucleotidesencoding the amino acid sequence of the predetermined polypeptide

The final polynucleotide from the plurality of test polynucleotides isoutputted as the final polynucleotide sequence to a user interfacedevice, a tangible computer readable storage medium; or the finalpolynucleotide is displayed in user readable form, where the finalpolynucleotide is deemed to be the test polynucleotide in the pluralityof test polynucleotides that has a fit that is closer to an ideal fitthan any other test polynucleotide in the plurality of testpolynucleotides.

In some embodiments, the plurality of sequence elements consists of upto five naturally occurring codons, of up to ten naturally occurringcodons, of up to fifteen naturally occurring codons, of up to twentynaturally occurring codons, of up to twenty-five naturally occurringcodons or up to thirty naturally occurring codons and the frequencylookup table places no restrictions on the frequency of occurrence of acodon in a test sequence when the codon is not in the frequency lookuptable.

In some embodiments, the global optimization algorithm is a heuristicsearch comprising a plurality of steps where, at each step in theplurality of steps, the global optimization algorithm comprises (i)quantifying a fit for each respective test polynucleotide in a subset ofthe plurality of test polynucleotides and (ii) enriching the pluralityof test polynucleotides by removing a portion of the subset of theplurality of test polynucleotides from the plurality of testpolynucleotides based on respective scores assigned to the testpolynucleotides in the subset of test polynucleotides. In someembodiments, at each step in the plurality of steps, the globaloptimization algorithm further comprises (iii) enriching the pluralityof test polynucleotides by adding a new subset of test polynucleotidesto the plurality of test polynucleotides, where each test polynucleotidein the new subset of test polynucleotides is derived from values forweights in one or more test polynucleotides already in the plurality oftest polynucleotides.

In some embodiments, the global optimization algorithm is a heuristicsearch. Examples of heuristic searches include, but are not limited toevolutionary algorithms, swarm-based optimization algorithms, memeticalgorithms, and differential evolution algorithms.

In some embodiments, the global optimization algorithm is a stochasticsearch. Examples of stochastic searches include, but are not limited to,simulated annealing algorithm, a directed monte-carlo samplingalgorithm, a stochastic tunneling algorithm, a parallel temperingalgorithm, a monte-carlo with minimization algorithm or a continuationmethod. See for example, Horst et al., 2000, Introduction to GlobalOptimization, Second Edition, Kluwer Academic Publishers; Neumaier,2004, Complete Search in Continuous Global Optimization and ConstraintSatisfaction, pp. 271-369 in: Acta Numerica 2004 (A. Iserles, ed.),Cambridge University Press; Mongeau et al., 2000, Optimization Methods &Software 13(3), pp. 203-226; Kirkpatrick et al., 1983, Science220:671-680, Hamacher, 2006, Europhys. Lett. 74(6):944, 2006; Hamacherand Wenzel, 1999, Landscape. Phys. Rev. E, 59(1):938-941; Wenzel andHamacher, 1999, Phys. Rev. Lett., 82(15):3003-3007; Hansmann, 1997,Chem. Phys. Lett., 281:140; Zhijun, November 1996, “The effective energytransformation scheme as a special continuation approach to globaloptimization with application to molecular conformation,” TechnicalReport, Argonne National Lab., IL (United States), November 1996, eachof which is hereby incorporated by reference herein in its entirety.

In some embodiments, each respective test polynucleotide in theplurality of the polynucleotides: (i) excludes a restriction sitesequence at a position in the respective test polynucleotide, (ii)incorporates a restriction site sequence at a position in the respectivetest polynucleotide, (iii) has a target G+C content, (iv) excludes asub-sequence, longer than an allowable length, that can be exactlyrepeated within either strand of the respective test polynucleotide, (v)excludes a first sub-sequence that can anneal at a greater than anallowable annealing temperature to a second sub-sequence within eitherstrand of the respective test polynucleotide, (vi) excludes of a hairpinturn, (vii) excludes a repeat element, (viii) excludes a ribosomebinding site, (ix) excludes a polyadenylation signal, (x) excludes asplice site, (xi) excludes an open reading frame in each possiblereading frame in the respective test polynucleotide other than thereading frame encoding the predetermined polypeptide, (xii) excludes apolynucleotide sequence that facilitates RNA degradation, (xiii)excludes an RNA polymerase termination signal, (xiv) excludes atranscriptional promoter, (xv) excludes an immunostimulatory sequence,(xvi) incorporates an immunostimulatory sequence, (xvii) excludes an RNAmethylation signal, (xviii) excludes a selenocysteine incorporationsignal, (xix) excludes an RNA editing sequence, (xx) excludes anRNAi-targeted sequence, (xxi) excludes an inverted repeat within thefirst x nucleotides encoding the amino acid sequence in the respectivetest polynucleotide, where x is any value between 1 nucleotide and onehundred nucleotides, or (xxii) excludes a DNA methylation signal.

In some embodiments, the fit determined for respective testpolynucleotide by the search of the mulitivariate space is furtherdetermined by (i) exclusion of restriction site sequence at a positionin the respective test polynucleotide, (ii) incorporation of arestriction site sequence at a position in the respective testpolynucleotide, (iii) presence of a target G+C content in the respectivetest polynucleotide, (iv) exclusion of a sub-sequence, longer than anallowable length, that can be exactly repeated within either strand ofthe respective test polynucleotide, (v) exclusion of a firstsub-sequence that can anneal at a greater than an allowable annealingtemperature to a second sub-sequence within either strand of therespective test polynucleotide, (vi) exclusion of a hairpin turn, (vii)exclusion of a repeat element, (viii) exclusion of a ribosome bindingsite, (ix) exclusion of a polyadenylation signal, (x) exclusion of asplice site, (xi) exclusion of an open reading frame in each possiblereading frame in the respective test polynucleotide other than thereading frame encoding the predetermined polypeptide, (xii) exclusion ofa polynucleotide sequence that facilitates RNA degradation, (xiii)exclusion of an RNA polymerase termination signal, (xiv) exclusion of atranscriptional promoter, (xv) exclusion of an immunostimulatorysequence, (xvi) incorporates an immunostimulatory sequence, (xvii)exclusion of an RNA methylation signal, (xviii) exclusion of aselenocysteine incorporation signal, (xix) exclusion of an RNA editingsequence, (xx) exclusion of an RNAi-targeted sequence, (xxi) exclusionof an inverted repeat within the first x nucleotides encoding the aminoacid sequence in the respective test polynucleotide, where x is between1 nucleotide and 100 nucleotides, or (xxii) exclusion of a DNAmethylation signal.

In some embodiments, the frequency lookup table further comprises amodel that computes an estimated expression property value for anexpression property of the predetermined polypeptide as a function of anucleotide sequence of a test polynucleotide, and the fit determined fora respective test polynucleotide in the search of the multivariate spaceis further determined by the estimated expression property value for thepredetermined polypeptide calculated by the model based on a nucleotidesequence of the respective test polynucleotide. In some embodiments, theexpression property is an estimate of a total amount of protein encodedby the respective test polynucleotide when expressed in the expressionsystem. In some embodiments, the expression property is an estimate of atotal amount of active protein encoded by the respective testpolynucleotide when expressed in the expression system. In someembodiments, the expression property is an estimate of a total amount ofsoluble protein encoded by the respective polynucleotide when expressedin the expression system.

In some embodiments, the sequence element in the plurality of sequenceelements consists of a codon. In some embodiments, a sequence element inthe plurality of sequence elements consists of a codon pair. A codonpair is a predetermined first codon and predetermined second codon thatare adjacent to each other and are in the same reading frame. In someembodiments, a sequence element in the one or more sequence elements isa nucleotide triplet in the +3 reading frame (encoding the “wobble” orthird base of one codon and the first two bases of the following codon).

In some embodiments, the plurality of sequence elements consists of upto five, up to ten, up to fifteen, up to twenty, up to twenty-five, orup to thirty naturally occurring codons and the frequency lookup tableplaces no restrictions on the frequency of occurrence of a codon in atest sequence when the codon is not in the frequency lookup table.

Another aspect provides a computer-readable medium storing one or morecomputer programs executable by a computer, the one or more computerprograms collectively comprising instructions for performing any of themethods described in this section. Another aspect provides an apparatuscomprising one or more processors and a memory, coupled to the one ormore processors, the memory storing one or more computer programs thatindividually or collectively comprise instructions for performing any ofthe methods disclosed in this section.

5.6.7 Use of Frequency Lookup Tables to Design a Polynucleotide Sequence

Another aspect of the present invention provides methods for designing apolynucleotide sequence that encodes a polypeptide sequence of apredetermined polypeptide. A frequency lookup table corresponding to anexpression system is obtained. The frequency lookup table comprises aplurality of sequence elements and a plurality of frequency ranges. Eachfrequency range in the plurality of frequency ranges is for acorresponding sequence element in the plurality of sequence elements.Each respective frequency range in the plurality of frequency rangesspecifies a range of frequencies with which the sequence elementcorresponding to the respective frequency range can occur in apolynucleotide that is to be expressed in the expression system. Eachrespective sequence element in the plurality of sequence elementsoptionally includes a frame designation which defines the frame of therespective sequence element relative to the reading frame of saidpredetermined polypeptide.

The polynucleotide sequence is defined using the frequency lookup table,where the defining comprises, for each respective sequence element inthe frequency lookup table, (i) determining whether the respectivesequence element encodes a portion of the polypeptide sequence in theframe designation specified for the respective sequence element whenthere is a frame designation for the respective sequence element in thefrequency lookup table and (ii) determining whether the respectivesequence element encodes a portion of the polypeptide sequence in anyframe when there is no a frame designation for the respective sequenceelement in the frequency lookup table. When the respective sequenceelement encodes a portion of the polypeptide sequence, the sequenceelement is incorporated into the polynucleotide at a frequency ofoccurrence that is within the frequency range specified for therespective sequence element in the frequency lookup table. Thepolynucleotide sequence is then outputted to a user interface device, atangible computer readable storage medium or the polynucleotide sequenceis displayed in user readable form.

In some embodiments, the portion of the polypeptide sequence is a firstamino acid that appears at a plurality of positions in the polypeptidesequence, a sequence element in the frequency lookup table consists of acodon that encodes the first amino acid, and the defining comprisesincorporating the codon for the amino acid in the polynucleotide at afirst frequency of occurrence at positions in the polynucleotide thatencode the amino acid at the plurality of positions in the polypeptidesequence, where the first frequency of occurrence is within thefrequency range specified for the sequence element in the frequencylookup table. For example, consider the case where the first amino acidis leucine and leucine appears at twenty positions in the polypeptidesequence. Further consider that the frequency lookup table contains asequence element that is the codon UUA for leucine in Homo sapiens and acorresponding frequency range that for the leucine that specifies thatthe codon is to be incorporated in a frequency range of 0.30 to 0.90relative to the five other codons that are synonymous for leucine inhomo sapiens (UUG, CUU, CUC, CUA, and CUG). This means that across thetwenty positions in the polypeptide sequence, the codon UUA is to beused between thirty percent (at six leucine positions) to ninety percent(at eighteen leucine positions) and the codons (UUG, CUU, CUC, CUA, andCUG) are to be used in the remaining positions.

In some embodiments, the portion of the polypeptide sequence in theframe designation specified for the respective sequence element is anamino acid pair that appears at a plurality of positions in thepolypeptide sequence, a sequence element in the frequency lookup tableconsists of a codon pair that encodes the first amino acid pair, and thedefining comprises incorporating the codon pair encoding the amino acidpair in the polynucleotide at a first frequency of occurrence atpositions in the polynucleotide that encode the amino acid pair at theplurality of positions in the polypeptide sequence, where the firstfrequency of occurrence is within the frequency range specified for thesequence element in the frequency lookup table.

In some embodiments, the frequency lookup table further comprises afrequency modification function for a predetermined sequence element,and the defining further comprises using the frequency modificationfunction to adjust, by an amount, the frequency range for thepredetermined sequence element where the frequency modification functionis determined by the frequency of occurrence, within the predeterminedpolypeptide sequence, of the portion of the polypeptide sequence encodedby the respective sequence element. Such a frequency modificationfunction is useful to provide tiered frequency ranges as a function ofthe number of times a sequence element appears within the predeterminedpolypeptide sequence. For example, if the sequence element appears lessthan a threshold number of times in the predetermined polypeptidesequence the frequency lookup table imposes a first frequency lookuptable and if the sequence element appears at or more than a thresholdnumber of times in the predetermined polypeptide sequence, the frequencylookup table imposes a second frequency lookup table. Exemplary valuesfor the threshold is any value between two and one thousand. In anotherembodiment, the frequency modification function operates on the maximumand/or the minimum of the frequency range for a predetermine sequenceelement based on a number of times the predetermined sequence elementappears in the predetermined polypeptide sequence. For example, theminimum of the frequency range can be a frequency that is divided by thenumber of times the predetermined sequence element appears in thepredetermined polypeptide sequence, and so forth. In some embodiments,the portion of the polypeptide sequence encoded by the respectivesequence element is a predetermined amino acid. In some embodiments, thesequence element comprises the wobble nucleotide of a first codon andthe first two nucleotides of a of a second codon, where the first andsecond codon respectively encode a first amino acid and a second aminoacid in a pair of adjacent amino acids in the polypeptide sequence,where the first codon is the 5′ codon and the second codon is the 3′codon in the pair of adjacent codons.

In some embodiments, the defining the polynucleotide sequence furthercomprises (i) exclusion of a restriction site sequence in thepolynucleotide sequence, (ii) incorporation of a restriction sitesequence in the polynucleotide sequence, (iii) a designation of a targetG+C content in the polynucleotide sequence, (iv) exclusion of asub-sequence, longer than an allowable length, that can be exactlyrepeated within either strand of the polynucleotide sequence, (v)exclusion of a first sub-sequence that can anneal at a greater than anallowable annealing temperature to a second sub-sequence within eitherstrand of the polynucleotide sequence, (vi) exclusion of a hairpin turnin the polynucleotide sequence, (vii) exclusion of a repeat element inthe polynucleotide sequence, (viii) exclusion of a ribosome binding sitein the polynucleotide sequence, (ix) exclusion of a polyadenylationsignal in the polynucleotide sequence, (x) exclusion of a splice site inthe polynucleotide sequence, (xi) exclusion of an open reading frame ineach possible reading frame in the test polynucleotide other than areading frame encoding the polypeptide sequence, (xii) exclusion of apolynucleotide sequence that facilitates RNA degradation in thepolynucleotide sequence, (xiii) exclusion of an RNA polymerasetermination signal in the polynucleotide sequence, (xiv) exclusion of atranscriptional promoter in the polynucleotide sequence, (xv) exclusionof an immunostimulatory sequence in the polynucleotide sequence, (xvi)incorporation of an immunostimulatory sequence in the polynucleotidesequence, (xvii) exclusion of an RNA methylation signal in thepolynucleotide sequence, (xviii) exclusion of a selenocysteineincorporation signal in the polynucleotide sequence, (xix) exclusion ofan RNA editing sequence in the polynucleotide sequence, (xx) exclusionof an RNAi-targeted sequence in the polynucleotide sequence, (xxi)exclusion of an inverted repeat within the first x nucleotides encodingthe amino acid sequence in the polynucleotide sequence, where x is anynumber between one nucleotide and one hundred nucleotides, and/or (xxii)exclusion of a DNA methylation signal in the polynucleotide sequence.

In some embodiments, the plurality of sequence elements comprises aplurality of codons, where the plurality of codons comprises all of thenatural codons encoding at least one amino acid, and where eachfrequency range for a codon in the plurality of codons comprises: amaximum percent occurrence of the corresponding codon relative to theoccurrence of all other codons that code the amino acid corresponding tothe corresponding codon throughout the polynucleotide sequence; and aminimum percent occurrence of the corresponding codon relative to theoccurrence of all other codons that code the amino acid corresponding tothe corresponding codon throughout the polynucleotide sequence, whereinthe minimum percent occurence of at least one codon in the plurality ofcodons is greater than zero percent and less than one hundred percent.

In some embodiments, the polypeptide sequence consists of each of aplurality of positions in the polypeptide sequence that contain the samepredetermined amino acid. In some embodiments, a sequence element in theplurality of sequence elements comprises a predetermined homopolymerstretch and a transcription pause site within a predetermined number ofnucleotides of each other.

Another embodiment provides a method of designing a polynucleotidesequence that encodes a polypeptide sequence of a predeterminedpolypeptide. The method comprises obtaining a first frequency lookuptable corresponding to an expression system, where the first frequencylookup table comprises a plurality of sequence elements and a firstplurality of frequency ranges, each frequency range in the firstplurality of frequency ranges is for a corresponding sequence element inthe plurality of sequence elements, each respective frequency range inthe first plurality of frequency ranges specifies a range of frequencieswith which the sequence element corresponding to the respectivefrequency range can occur in the polynucleotide sequence and eachrespective sequence element in the plurality of sequence elementsoptionally includes a frame designation which defines the frame of therespective sequence element relative to the reading frame of saidpredetermined polypeptide.

The method further comprises obtaining a second frequency lookup tablecorresponding to the expression system, where the second frequencylookup table comprises the plurality of sequence elements and a secondplurality of frequency ranges, each frequency range in the secondplurality of frequency ranges is for a corresponding sequence element inthe plurality of sequence elements, each respective frequency range inthe second plurality of frequency ranges specifies a range offrequencies with which the sequence element corresponding to therespective frequency range can occur in the predetermined polypeptide,and each respective sequence element in the second plurality of sequenceelements optionally includes a frame designation which defines the frameof the respective sequence element relative to the reading frame of saidpredetermined polypeptide.

The polynucleotide sequence is defined. This defining comprises, foreach respective sequence element in the plurality of sequence elements,determining a number of times an amino acid encoded by the sequenceelement is present in the polypeptide, where, when the number of timesis more than a threshold amount the sequence element is incorporatedinto the polynucleotide at a frequency of occurrence that is within thefrequency range specified for the respective sequence element in thefirst frequency lookup table. When the number of times is equal to orless than the threshold amount, the sequence element is incorporatedinto the polynucleotide at a frequency of occurrence that is within thefrequency range specified for the respective sequence element in thesecond frequency lookup table. The polynucleotide sequence is thenoutputted to a user interface device, a tangible computer readablestorage medium. Or the polynucleotide sequence is displayed in userreadable form.

Another aspect provides a method of designing a polynucleotide sequencethat encodes a polypeptide sequence of a predetermined polypeptide. Themethod comprises obtaining a first frequency lookup table correspondingto an expression system. The first frequency lookup table comprises aplurality of sequence elements and a first plurality of frequencyranges. Each frequency range in the first plurality of frequency rangesis for a corresponding sequence element in the plurality of sequenceelements. Each respective frequency range in the first plurality offrequency ranges specifies a range of frequencies with which thesequence element corresponding to the respective frequency range canoccur in the polynucleotide sequence. Each respective sequence elementin the plurality of sequence elements optionally includes a framedesignation which defines the frame of the respective sequence elementrelative to the reading frame of said predetermined polypeptide. Asecond frequency lookup table corresponding to the expression system isobtained. The second frequency lookup table comprises the plurality ofsequence elements and a second plurality of frequency ranges. Eachfrequency range in the second plurality of frequency ranges is for acorresponding sequence element in the plurality of sequence element.Each respective frequency range in the second plurality of frequencyranges specifies a range of frequencies with which the sequence elementcorresponding to the respective frequency range can occur in saidpredetermined polypeptide. Each respective sequence element in thesecond plurality of sequence elements optionally includes a framedesignation which defines the frame of the respective sequence elementrelative to the reading frame of said predetermined polypeptide. Thepolynucleotide sequence is defined. This defining comprises, for eachrespective sequence element in the plurality of sequence elements,determining a frequency with which an amino acid encodable by thesequence element is present in the polypeptide relative to the totalnumber of amino acids in the polypeptide where, when the frequency ismore than a threshold amount the sequence element is incorporated intothe polynucleotide at a frequency of occurrence that is within thefrequency range specified for the respective sequence element in thefirst frequency lookup table, and when the frequency is equal to or lessthan the threshold amount, the sequence element is incorporated into thepolynucleotide at a frequency of occurrence that is within the frequencyrange specified for the respective sequence element in the secondfrequency lookup table. The polynucleotide sequence is outputted to auser interface device, a tangible computer readable storage medium.Alternatively, the polynucleotide sequence is displayed in user readableform.

Another aspect provides a method of designing a polynucleotide sequencethat encodes a polypeptide sequence of a predetermined polypeptide. Themethod comprises obtaining a plurality of frequency lookup tables (e.g.,two or more frequency lookup tables, three or more frequency lookuptables, four or more frequency lookup tables, five or more frequencylookup tables, or between two and twenty frequency lookup tables, etc.)each frequency lookup table in the plurality of frequency lookup tablescorresponding to the same expression system, where each respectivefrequency lookup table in the plurality of frequency lookup tablescomprises: (i) a plurality of sequence elements and a plurality offrequency ranges, (ii) each frequency range in the plurality offrequency ranges is for a corresponding sequence element in theplurality of sequence elements, (iii) each respective frequency range inthe plurality of frequency ranges specifies a range of frequencies withwhich the sequence element corresponding to the respective frequencyrange can occur in the polynucleotide sequence, (iv) each respectivesequence element in the plurality of sequence elements optionallyincludes a frame designation which defines the frame of the respectivesequence element relative to the reading frame of said predeterminedpolypeptide; and (v) a unique frequency lookup table conditioncriterion. In the method, the polynucleotide sequence is defined. Thedefining comprises, for each respective sequence element in theplurality of sequence elements, determining a frequency lookup tablecondition for the respective sequence element in the polynucleotidesequence, where, when the frequency lookup table condition satisfies aunique frequency lookup table condition criterion for a correspondingfrequency lookup table in the plurality of frequency lookup tables, thesequence element is incorporated into the polynucleotide at a frequencyof occurrence that is within the frequency range specified for therespective sequence element in the corresponding frequency lookup table.The method further comprises outputting the polynucleotide sequence to auser interface device, a tangible computer readable storage medium ordisplaying the polynucleotide sequence in user readable form. In someembodiments, the unique frequency lookup table condition criterion forthe respective sequence element is a number of times an amino acidencoded by the sequence element is present in the polypeptide.

Another aspect provides a computer-readable medium storing one or morecomputer programs executable by a computer, the one or more computerprograms collectively comprising instructions for performing any of themethods described in this section. Another aspect provides an apparatuscomprising one or more processors and a memory, coupled to the one ormore processors, the memory storing one or more computer programs thatindividually or collectively comprise instructions for performing any ofthe methods disclosed in this section.

5.7 RELATIVE AND ABSOLUTE CODON FREQUENCIES

In some cases, the level of protein expression, the level of solubleprotein expression or the level of active protein expression obtainedfrom a polynucleotide in an expression host may depend upon the relativefrequencies with which different synonymous codons are used within thepolynucleotide. This is the information that is typically encoded in acodon bias table. In some cases, the level of protein expression, thelevel of soluble protein expression or the level of active proteinexpression obtained from a polynucleotide in an expression host maydepend upon the absolute frequencies with which different synonymouscodons are used within the polynucleotide. This is different from theinformation typically encoded in a codon bias table, since it dependsnot only upon the polynucleotide but also upon the encoded polypeptide.

As an example to clarify this difference between codon bias or relativecodon frequency and absolute codon frequency, consider a polypeptidecontaining 100 amino acids of which 20 are Tyr. This polypeptide couldbe encoded by a polynucleotide that used the TAT codon 7 times (i.e. 35%of 20) and the TAC codon 13 times (i.e. 65% of 20). Then consider asecond polypeptide containing 100 amino acids of which 40 are Tyr. Thissecond polypeptide could be encoded by a second polynucleotide that usedthe TAT codon 14 times (i.e. 35% of 40) and the TAC codon 26 times (i.e.65% of 40). Both the first and second polynucleotides have codon biasesfor TAT of 0.35 and for TAC of 0.65. That is the codon TAT is used toencode Tyr 35% or 0.35 of the time, and the codon TAC is used to encodeTyr 65% or 0.65 of the time. However the first polynucleotide uses TATat an absolute frequency of 7:100 (or 0.07), that is the codon TAToccurs 7 times per hundred codons in the polynucleotide. The secondpolynucleotide uses TAT at an absolute frequency of 14:100 (or 0.14),because the codon TAT occurs 14 times per hundred codons in thepolynucleotide. The codon bias or relative codon frequency is thus thefrequency of a codon relative to all synonymous codons, while theabsolute codon frequency is the frequency of a codon relative to bothsynonymous and non-synonymous codons, that is to the total number ofcodons used to encode the polypeptide in the polynucleotide.

5.8 DESIGN OF CODON FUNCTION TABLES

In some cases, the level of protein expression, the level of solubleprotein expression or the level of active protein expression obtainedfrom a polynucleotide in an expression host may depend upon the rate atwhich the polynucleotide is translated, which is also the rate at whichthe polypeptide is extended. In some embodiments of the invention, forthe synthesis of some polypeptides in some expression systems thisrelationship may be direct: the more quickly a ribosome is able to addall of the amino acids in a polypeptide, the more polypeptides may beproduced per unit of time and so the greater the expression levels ofpolypeptide that may be obtained. In some embodiments of the invention,for other polypeptides and other expression systems it may be an inverserelationship: the more slowly a ribosome is able to add all of the aminoacids in a polypeptide, the more time the polypeptide may have to fold,and so the more soluble protein may be produced. In some embodiments ofthe invention it may be advantageous to design a polynucleotide thatallows amino acids to be added most rapidly to the polypeptide except atcertain positions, regions, or domains of the polypeptide structurewhere it is critical to slow translation to promote proper folding. Suchpositions or regions may be at or near protein structural domainboundaries or consist of multiple contiguous or non-contiguous aminoacids within a structural domain where folding is sensitive totranslation rate.

One factor that affects the rate of translation in some expressionsystems under some conditions is the rate with which each charged tRNAbinds at the A site of the ribosome; this step is followed byrecognition of the cognate codon bringing the aminoacyl stem of the tRNAinto the A site of the ribosomal large subunit, joining of the incomingamino acid to the growing peptide chain by a peptidyl transferaseactivity, and translocation of the tRNA and peptide chain to theribosome's P site. The time taken for addition of an amino acid to agrowing peptide chain can thus be approximated ast _(ad) =t _(c) +t _(ε)where

t_(ad) is the time taken for addition of an amino acid to the peptidechain,

t_(c) is the time taken for the correct charged tRNA to bind to itscodon and fully occupy the ribosome's A site, and

t_(e) is the time taken for the catalytic and translocation steps.

The time taken for a single ribosome to complete synthesis of an entirepolypeptide can therefore be approximated as:t _(syn) =t _(in) +Σt _(ad)where

t_(syn) is the time taken to synthesize the polypeptide,

t_(in) is the time taken for the ribosome to bind to the message andinitiate translation.

Σt_(ad) is the sum of the times taken for the addition of each aminoacid.

In selecting preferred codons, the rate of translational initiation maybe considered independent or dependent on the polypeptide codingsequence. The degree to which initiation is dependent on the codingsequence may be influenced by the initial peptide sequence, the hostorganism, and the expression system employed. In systems whereinitiation depends primarily upon sequences to the 5′ of the openreading frame, it is advantageous to simplify the problem by consideringthat the time taken to initiate translation is approximately constantfor all possible codon sequences. In other systems it is advantageous toconsider that both initiation and elongation times are dependent oncodon selection, and make adjustments to sequences that may affectinitiation. Such sequences may include those that promote secondarystructure of the mRNA near or possibly covering the start codon and/orthe ribosome binding site (RBS), and thus may hinder proper initiation.To minimize interference with initiation, a polynucleotide can bedesigned by choosing codons that result in an initial coding sequencethat does not interfere with ribosome binding. Optimal initial sequencesmay be highly dependent on the sequence of the 5′ UTR of the mRNA,particularly that of and near the RBS. Such designs can be performed bymRNA secondary structure prediction software to analyze RBSinterference.

Ribosomes are especially susceptible to abortion of elongation in thefirst 15-20 codons translated. Use of rapidly translated codons in theinitial coding sequence can minimize abortion as well as increasing theinitiation rate by clearing the ribosome binding site faster. Preferredcodons for the initial coding sequence may be identified bysystematically varying the codons for the first 10, 20 or 30 amino acidswhile keeping the remainder of the coding sequence constant, or whilevarying it independently of the variation in the first 10, 20 or 30codons. Experimental expression data can then be used to distinguishcorrelations between initial sequence and expression level and optimalsequences can then be determined.

To design polynucleotide sequences that will result in shorter synthesistimes for an encoded polypeptide, selecting codons that result in lowervalues for Σt_(ad) may be advantageous. The speed with which theincoming amino acid is joined to the growing peptide chain by a peptidyltransferase activity and the tRNA and peptide chain are translocated tothe ribosome's P site do not depend to a significant degree upon thecodon used. Thus shorter times for the synthesis of a polypeptide willresult from shorter times taken for a charged tRNA to bind to its codonon the mRNA and fully occupy the A site of the ribosome.

The rate at which charged tRNA binds to and is selected by the ribosomefor A-site entry may depend on the concentration of charged tRNAavailable in the expression system. The higher the concentration theshorter the time expected for the tRNA to associate with its codon atthe A-site. The overall tRNA selection rate may also depend on the rateat which tRNA dissociates from the A-site before recognition of thecorrect codon-anticodon pairing and selection for petidyl transfer.Multiple tRNA species may recognize a particular codon and a particulartRNA species often may recognize multiple codons. The tRNA-codonassociation and dissociation rates may depend on the particular tRNAspecies and codon involved. These rates may also be influenced by thetRNA and codon occupying the P-site of the ribosome, either by directphysical interaction of the P-site tRNA with the closely binding A-sitetRNA or by influencing the presentation of the codon to be recognized. Afurther influence on tRNA selection may be the rate at which theribosome recognizes the correct codon-anticodon pairing and allows entryof the tRNA to the A-site of the large ribosomal subunit for peptidyltransfer. This rate may depend on the specific tRNA species and codoninvolved and also may depend on the P-site tRNA and codon if theseinfluence how the ribosome recognizes the codon-anticodon interaction.

The process by which a charged tRNA is selected by the ribosome forpeptidyl transfer may be described by the following simplifiedexpression:P_(n)R+T_(c2)

P_(n)RT_(C2)*→P_(n)RT_(C2)→P_(n+1)R+T_(U1)

where

-   -   P_(n)R is the complex of the P-site tRNA with the mRNA        programmed ribosome,    -   T_(C2) is the aminoacyl-tRNA to be selected (ternary complex of        EF-Tu and the charged tRNA),    -   P_(n)RT_(C2)* is the initial complex of the aminoacyl-tRNA with        the ribosome,    -   P_(n)RT_(C2) is the complex after recognition of the        codon-anticodon interaction and full entry of the aminoacyl-tRNA        into the ribosomal A-site,    -   P_(n+1)R is the ribosomal complex after peptidyl transfer and        translocation of the A-site tRNA to the P-site, and    -   T_(u1) is the deacylated form of the initial P-site tRNA.

In selecting codons for shorter translation times, one may choose toassume that the tRNA selection rate is dependent primarily on the rateat which the cognate charged tRNA associates with the ribosome. Underthis assumption the rate of selection would be highly dependent on thecharged tRNA ternary complex concentration. Each tRNA exists in a fullternary complex (will be referred to simply as “charged”) or in someother form inactive for peptide acceptor use by the ribosome (will bereferred to as “uncharged”). The balance of tRNA species i may beexpressedT _(Ti) =T _(Ci) +T _(Ui)where

T_(Ti) is the total tRNA of species i,

T_(Ci) is the concentration of charged tRNA of species i, and T_(Ui) isthe concentration of uncharged tRNA of species i,

The rate at which charged tRNA is consumed may be expressed

where k_(Ui) is an overall rate constant for loss of charged tRNAspecies i, which includes consumption for protein synthesis, chemicaldeacylation and any other form of reversible loss of active ternarycomplex. The term k_(Ci) is the rate constant for charging of tRNAspecies i. In determining the relationship of codon usage to expressionrate, one simplification could be to assume that T_(Ci) is in steadystate during protein synthesis and that total tRNA of species i isconstant. If irreversible degradation of T_(Ci) is assumed to beinsignificant, then the steady-state assumptions yield the relationship:T _(Ci) =k _(Ci) T _(Ui) /k _(Ui)

And substituting T_(Ui) with (T_(Ti)−T_(Ci))

$T_{Ci} = \frac{T_{Ti}\; k_{Ci}}{k_{Ui} + k_{Ci}}$

The overall rate constant of uncharging may be considered the sum of theconstants for consumption by protein synthesis, k_(Pi), and all otherreversible degradation, k_(Di):k _(Ui) =k _(pi) +k _(Di)

If consumption for protein synthesis is considered the dominant path ofcharged tRNA loss, the expression for charged tRNA level may besimplified to:

$T_{Ci} = \frac{T_{Ti}\; k_{Ci}}{k_{Pi} + k_{Ci}}$

The first order rate constant for consumption of tRNA species i inprotein synthesis can be expressed as the product of the concentrationof a cognate codon j at the ribosomal A-site, C_(j), times the rateconstant for reaction with that codon, k_(Sj), summed for all codonsread by that tRNA:k_(pi)=Σk_(Sj)C_(j)

Thus, charged tRNA may be expressed as follows:

$T_{Ci} = \frac{T_{Ti}\; k_{Ci}}{{\sum\limits^{\;}\;{k_{sj}\; C_{j}}} + k_{Ci}}$

The above equation shows one way to understand charged tRNA levels as afunction of codon usage. As consumption, Σk_(S) _(j) C_(j), becomessignificant relative to charging, k_(Ci), the level of charged tRNA islowered, potentially lowering the rate of protein synthesis at stepsinvolving the lowered tRNA species. One way specific tRNA consumptionmay be increased is by increasing the frequency usage of codons read bythe tRNA, raising C_(j).

One way the translation rate of a particular codon v_(Cj) read by tRNAspecies i may be modeled is the product of the charged tRNAconcentration and the reaction rate constant for the codon, summed forall tRNA species—codon combinations for that codon:v_(Cj)=ΣT_(Ci)k_(Sj)

The complete relationship may be considerably more complicated due tocompetition between tRNAs for the ribosomal A-site and other factors,although the above approximation may be sufficient for practicalmodeling of the relationship between codon usage and expression level.In this relationship it can be seen that codon translation rate isincreased by higher steady-state charged tRNA level and by a higher rateconstant for translation of the codon. In choosing codons to maximizeexpression, one approach according to the above discussion would be touse codons that are read at a high rate, but use a balance of codonsread by different tRNAs such that specific tRNA consumption remains lowrelative to recharging and T_(Ci) remains high as possible. This balancewill depend upon the frequency of each amino acid within thepolypeptide. For example in a polynucleotide encoding a polypeptide withmany leucine residues but few serine residues it will be important tobalance the codons for leucine but less important to balance the codonsfor serine. Similarly it will generally be more important to balance thecodons for other highly represented amino acids within an encodedpolypeptide than those that occur infrequently within the polypeptide.Thus in some embodiments it is advantageous to use different codon biastable or lookup tables or design algorithms may be used for designingpolynucleotides to encode different polypeptides.

Context of the codon in the mRNA, such as the nature of the precedingcodon, the position within the open reading frame, or surroundingstructure in the mRNA, may alter the rate of reaction of a tRNA with acodon. In a more generalized form, therefore, k_(Sj) would refer to thereaction rate with a cognate codon in a context j and Σk_(S) _(j) C_(j)would be the sum for all contexts and rates for cognate codons for thetRNA species. One way codon context may be simplified is by consideringonly neighboring sequence in the mRNA. For example, one may include inthe context with a codon only the preceding codon (61 possible contextsfor each codon after the start codon), only the preceding dinucleotide(16 possible contexts), or only the preceding nucleotide (4 possiblecontexts).

In determining the relationship between gene sequence and expressionlevel, the above equations suggest alternative ways to define theparameters for system modeling. In one preferred embodiment of theinvention, frequencies of individual codons may be treated asindependent variables. In another preferred embodiment, relationshipsbetween codon usage and tRNA concentration are used, as exemplifiedabove, to determine expression models that may more accurately reflectthe mechanism of protein translation. In such models, expression is fitas a function of the influence of codon usage on tRNA levels and therate constants for codon translation. For example, protein synthesis maybe modeled as proportional to charged tRNA levels and related to codonusage and recharging rates as described above. In another embodiment,codon context information may be added to further refine the model byaccounting for any influence of context on translation rates asdiscussed above.

5.9 DESIGN OF POLYNUCLEOTIDES FOR IMPROVED EXPRESSION PROPERTIES OFENCODED POLYPEPTIDES

Measuring one or more expression properties of the polynucleotides in acodon variant set produces a set of correlated values for sequenceelement usage and one or more expression property. Sequence elementusage can then be taken directly from one of the best expressingvariants. Alternatively sequence element usage can be calculated as anaverage value or a weighted average from a set of the best expressingvariants. Alternatively sequence-expression modeling can produce singlevalues or ranges of values for the absolute or relative frequency foreach sequence element that best correlates with desired expressionproperties. These frequencies can be provided as a frequency lookuptable or a codon frequency table or matrix, or a codon lookup table. Insome embodiments, these tables or matrices provide a target value orrange of values for the frequencies of codons for one or more aminoacids that should be used in designing a polynucleotide to encode apolypeptide.

Frequency lookup tables can be created from sequence-expressionrelationships to describe a target range of relative or absolutefrequencies for each sequence element instead of the single value foreach codon that is found in codon bias tables that describe thefrequencies of codons found in naturally occurring sequences or genomes.These frequency lookup tables can thus provide a target range for thefrequency of each sequence element (e.g. codon) in a designedpolynucleotide, rather than a single target value. This is particularlyadvantageous when the frequencies of some sequence elements have strongcorrelations with desired expression properties and the frequencies ofother sequence elements appear to be less important.

This balance will depend upon the frequency of each amino acid withinthe polypeptide. For example in a polynucleotide encoding a polypeptidewith many leucine residues but few serine residues it will be importantto balance the codons for leucine but less important to balance thecodons for serine. Similarly it will generally be more important tobalance the codons for other highly represented amino acids within anencoded polypeptide than those that occur infrequently within thepolypeptide. Thus different frequency lookup tables or design algorithmsmay be used for designing polynucleotides to encode differentpolypeptides.

One codon variant set can be expressed in more than one expressionsystem, sequence-expression relationships can be derived from more thanone expression system and for more than one expression property. In someembodiments, polynucleotides are designed using sequence element targetfrequencies expression derived from measuring expression properties ofthe codon variant set. It is advantageous to express these designedpolynucleotides in the same expression system that was used forexpression of the codon variant set, since different expression systemsor conditions may differ in the factors that are rate limiting for theexpression property of interest.

Another aspect of the present invention provides a computer programproduct for use in conjunction with a computer system, the computerprogram product comprising a computer readable storage medium and acomputer program mechanism embedded therein. The computer programmechanism comprising instructions for carrying out any step of anymethod disclosed herein that does not involve expressing a protein ormeasuring an abundance of a protein. Still another aspect of theinvention provides a computer system comprising a central processingunit and a memory, coupled to the central processing unit, the memorystoring the aforementioned computer program product.

6. EXAMPLES

The following examples are set forth so as to provide those of ordinaryskill in the art with a complete description of how to make and useembodiments of the present invention, and are not intended to limit thescope of what is regarded as the invention.

6.1 DESIGN, SYNTHESIS & EXPRESSION TESTING OF A CODON VARIANT SET FORPH129 DNA POLYMERASE 6.1.1 Ph±29 Variant Design

An expression construct for testing codon variants of the 1.7 kb genefor the DNA polymerase of the Bacillus sp. phage phi29 was designed tocontain sequence elements required for expression in an E. coli hostcell. To the 5′ of the open reading frame were a T7 promoter andribosome binding site (sequences provided as SEQAA), to the 3′ of thephi29 open reading frame were two stop codons and a T7 terminator(sequences provided as SEQAB). SEQAA also contains other importantvector sequences: an origin of replication, a gene conferring resistanceto the antibiotic kanamycin, and a gene expressing the lac repressor.The sequences SEQAA and SEQAB were identical for all phi29 DNApolymerase variants tested, the only differences were in the codonsselected to encode the polypeptide in the open reading frames, and allof these DNA sequences resulted in identical polypeptides.

The natural coding sequence for phi29 polymerase was obtained fromGenBank and used as a template for gene design. A set of seven genedesign parameters was varied between the designs. These were:

1. Codon bias. Codon bias describes the relative frequency with whichsynonymous codons are used to encode an amino acid. We used two codonbias tables, one calculated from all of the genes in the E. coli genome(‘E. coli’ table) and a second from a subset of 27 genes that are highlyexpressed (‘E. coli II’ table) (Gustafsson et al., 2004, “Codon bias andheterologous protein expression,” Trends Biotechnol 22, 346-53, Henautand Danchin, 1996, “Analysis and predictions from Escherichia colisequences,” Escherichia coli and Salmonella typhimurium Cellular andMolecular Biology 2, 2047-2066, Sharp and Li, 1987, “The codonAdaptation Index—a measure of directional synonymous codon usage bias,and its potential applications,” Nucleic Acids Res 15, 1281-95). Todesign each gene variant encoding phi29 polymerase, we used GeneDesigner software (Villalobos et al., 2006, “Gene Designer: a syntheticbiology tool for constructing artificial DNA segments,” BMCBioinformatics 7, 285). For each consecutive amino acid the programchooses a codon with a probability that is proportional to its frequencyin the chosen bias table. Thus the overall bias of the gene approximatesthat of the bias table.

2. Cutoff threshold. Several previous reports have shown that use ofsome rarely used codons may lower expression, particularly when theseare used frequently or in tandem (Kane, 1995, “Effects of rare codonclusters on high-level expression of heterologous proteins inEscherichia coli,” Curr Opin Biotechnol 6, 494-500, Shu et al., 2006,“Inhibition of translation by consecutive rare leucine codons in E.coli: absence of effect of varying mRNA stability,” Gene Expr: 13,97-106). We therefore used Gene Designer to exclude codons whosefrequencies in the codon bias table are lower than a selected threshold(the cutoff threshold). We varied this threshold to test whetherinclusion of codons used at intermediate frequency or occasionalinclusion of rare codons have a detrimental effect. At the highestthreshold settings used (25%, that is, the only codons allowed are thosethat are used to encode an amino acid more than 25% of the time in thegenes from which that codon bias table is derived), 5-6 amino acids thatcould be encoded by multiple codons are restricted to a single choice.At the lowest settings (2%) only 0-6 of the rarest codons are excluded.

3. Internal RNA structure. After initial coding we analyzed the RNAsecondary structure of each variant using UNAFold (Markham and Zuker,2005, “DINAMelt web server for nucleic acid melting prediction,” NucleicAcids Res. 33, W577-W581). RNA structures were determined for every 50nucleotide window of the coding sequence (1676 windows within the 1725nucleotide open reading frame), and for the 3′ terminal tail of the mRNAconsisting of the last 50 nucleotides of the coding sequence through tothe expected terminus. A 50 nucleotide window was used because it shouldidentify local structures that can form between consecutive ribosomes onan mRNA: ribosome spacing is estimated at 50 nucleotides for a rapidlytranslating message. One region of the mRNA, the 50 nt window fromposition +147 to +196, was found to be especially prone to formingstrong hairpin structures (FIG. 1). This structure was manually modifiedin selected genes by minimal codon substitutions, within the bias andthreshold limits.

4. RNA structure at the 5 initiation site. We used UNAFold to calculatethe RNA structure for the first 121 nucleotides from the 5′ end of themRNA to position +50 (FIG. 1). This structure was manually modified inselected genes by minimal codon substitutions, within the bias andthreshold limits.

5. 5′AT wobble. We manually modified the percentage of the first 15codons that use A or T in the 3d position.

6. GC runs. Runs of G and/or C of 6 nucleotides or more were avoided insome sequences by defining SSSSSS(S=G or C) as a motif to avoid in GeneDesigner.

7. Identity to wild type. We selected sequences with different degreesof identity to the wild type. This was performed using Gene Designer.

In addition to the codon bias table and threshold parameterssystematically varied, the covariant gene codon adaptation index (CAI)(Sharp and Li, 1987, “The codon Adaptation Index—a measure ofdirectional synonymous codon usage bias, and its potentialapplications,” Nucleic Acids Res 15, 1281-1295), the parameter mostcommonly used for gene optimization, was monitored in the dataset andincluded in modeling. The adaptiveness of a codon is defined as itsusage frequency relative to that of the most frequently used synonymouscodon in the E. coli II bias table. The CAI for the gene is the averagecodon adaptiveness over all positions. A CAI of 1 means only the mostfrequent codon is used for all 20 amino acids at every position.

To assess the impact of these seven parameters on protein expression,each was varied within practical limits. Five parameters (codon bias,threshold, internal RNA structure, AT wobble, and GC runs), weresystematically varied among variants 1-8. Two parameters, 5′-RNA andidentity to wild type, were difficult to systematically vary along withthe remaining set of variables, so a few genes were constructed for eachwhere only codon bias was varied and the other parameters were notcontrolled. The design properties of the first 21 variants are shown inFIG. 2. On average the pairwise sequence identities of the variants was82% and no two are more similar than 91%, except 16 and 17 (97%) and 19and 20 (93%).

6.1.2 Phi 29 Variant Synthesis and Cloning

The gene variants were all synthesized by standard gene synthesisprocedures and cloned into a pET24a expression vector between the XbaIand EcoRI restriction sites. This put each gene under transcriptionalcontrol of the strong T7 RNA polymerase promoter of this vector. Eachfinal construct was completely sequenced in both directions to becertain that the complete sequence was as designed. The plasmid was thenused to transform E. coli expression host strain BL21(DE3) harboring asecond plasmid for low level expression of T7 lysozyme, pLysS. Thebackground expression of T7 lysozyme, an inhibitor of T7 RNA polymerase,gives tight repression of heterologous expression prior to induction tominimize potential gene toxicity which could affect data quality.

6.1.3 Phi 29 Variant Protein Expression

Proteins were expressed from the variant genes using standard methods.Prior to analysis of the variants, expression was analyzed for multiplevariants to determine appropriate expression time and temperature.Strong, consistent expression was achieved at 30° C., a commonly usedtemperature for heterologous expression in E. coli. Time courses at 30°C. showed expressed protein levels increasing to a maximum afterapproximately two hours, as the cells entered stationary phase growth,and remaining steady for at least five hours. Relative proteinexpression levels between variants were consistent throughout the timecourse. For our variant analysis we chose to express for four hours at30° C.

For each variant, three transformants were picked and cultured overnightin 2 ml Luria Broth (LB) containing appropriate antibiotics to maintainthe expression vector and pLysS plasmid. The overnight cultures werediluted 50-fold in fresh media and incubated at 37° C. until the cellswere in mid-log growth (OD at 600 nm ˜0.6). Expression was induced byaddition of IPTG to 1 mM and incubation for four hours at 30° C. Finaloptical densities of cultures were measured and equivalent amounts ofculture were analyzed by polyacrylamide gel electrophoresis (a sampleset of variant expression is shown in FIG. 3). Gels were stained withSypro Ruby (Pierce), visualized by fluorescence imaging, and proteinband intensities quantified using TotalLab 100 image analysis software(Nonlinear Dynamics, Inc). Each gel contained protein concentrationstandards to calibrate band intensity. Three replicates of a referenceclone (a phi29 polymerase variant identical to variant 21 but containingtwo point mutations in the 5′ untranslated region) were expressed inparallel cultures in each experiment to allow corrections to be made forany experiment to experiment variation. Reported expression levels areall relative to this reference. The detection limit of the assay wasapproximately 1 μg of phi29 polymerase per ml culture at an A₆₀₀=3. Thisis ˜5% of the reference variant expression level. The standard error ofmeasured expression for variant repeats was generally <20% of the mean.

Quantitation of expression levels for the first 21 variants synthesizedare shown in FIG. 2. There are a wide range of expression levels amongthe variants, demonstrating a good sampling of expression relevantfeatures in our gene designs. Levels ranged from below detection to 3times the reference, a range of two orders of magnitude.

6.1.4 Analysis of Gene Design Parameters for Phi 29 Variants

To determine the influence of the various gene design parameters onexpression, the data in FIG. 2 was fit to partial least squares (PLS)models using PLS Toolbox 4.0 software (Eigenvector, Inc.) within MATLAB(Mathworks, Inc). PLS regression is a highly reliable method formodeling systems data where the number of independent variables is high,approaching or even exceeding the number of samples (Eriksson et al.,2004, “Using chemometrics for navigating in the large datasets ofgenomics, proteomics, and metabonomics (gpm),” Anal Bioanal Chem 380,419-29). In PLS regression, multivariate data are transformed to avariable space with reduced dimensionality that is defined by neworthogonal variables (latent variables). The latent variables are linearcombinations of the original variables, calculated to maximizecorrelation between independent and dependent variables in as fewdimensions as possible. Cross-validation methods are used to determinethe optimal number of latent variables to use in the model, so that fitbetween data and model is maximized without over-fitting.

Correlation between observed and predicted expression levels for aninitial PLS model of the phi29 polymerase variants data is shown in FIG.4 part A. For this model, all variables listed in FIG. 2 were included,except GC %, which tightly correlates with ‘identity to wt’ and onlycontributes noise to this model fit. For modeling, the codon bias tableis described by a binary term with E. coli genomic bias assigned thevalue −1 and E. Coli II bias is +1. RNA structure variables are enteredas the absolute value of the predicted minimal free energy fold. In thismodel, 65% of the independent variables contributing to variation inprotein expression were captured in latent variable 1 (LV1) and anadditional 14% in LV2. FIG. 4 part B shows the loads of each of theinitial design parameters on the first latent variable (LV1). This is ameasure of the extent to which each initial design parameter influencesthe expression level of the phi29 gene variants.

The degree of identity between a variant and the wild type sequence wasthe most dominant factor in determining the protein expression levelsthat could be obtained from a gene. The two variants most different fromwild-type, 19 and 20, showed more than two-fold higher expression thanthe next best variant. Also, contrary to conventional thinking,increasing the cutoff threshold for codons correlated with lowerexpression levels. A second parameter, the codon adaptation index (CAI),was also considered. As defined here, the adaptiveness of a codon isdefined as its usage frequency relative to that of the most frequentlyused synonymous codon in the E. coli II bias table. The CAI for the geneis the average codon adaptiveness over all positions. A commonly usedgene optimization method is to maximize CAI, although no significantcorrelation of CAI with heterologous expression has been adequatelydemonstrated. CAI correlated negatively with protein expression in ourexperiments (FIG. 4 part B). High CAI scores and high cutoff thresholdsboth reflect a reduction in the diversity of codons used for some aminoacids. These results suggest that limiting the total number of differentcodons used in the gene, may be detrimental. The model also suggests anegative effect of A/T bias in the third codon position of the first 15codons and a positive effect of 5′RNA structure. It is not clear whystronger RNA structure around the initiation AUG would have a positiveeffect.

6.1.5 Analysis of Codon Bias and Expression for Phi 29 Variants

The factors affecting expression of phi29 variants all point to theinfluence of codon usage on expression. Two of the possible ways inwhich codon usage can be important are that there are relatively smallnumbers of rate-limiting positions, or that there is an optimal codonbias that is different from the E. coli genomic or Class II biases thatwe used in our original designs.

A new partial least squares (PLS) models was constructed using PLSToolbox 4.0 software (Eigenvector, Inc.) within MATLAB (Mathworks, Inc),analyzing the sequences and expression levels of phi29 variants shown inFIG. 2 to identify specific codon choices that correlate best withactivity. The variables used were the possible codons used at eachposition. For each codon position in the gene, the possible codonchoices were assigned the value 1 if used and 0 if not, as shown for thefirst few positions in FIG. 5.

The complete set of variants contains 1517 variables. Figure F part Ashows the fit between predicted and measured expression levels for aPartial Least Squares (PLS) model of the expression and sequence of the21 variants described in FIG. 2. Figure F part B shows the positions inthe polynucleotide sequence that were identified in this model aspossible expression determining positions or regions.

PLS analysis of gene design features indicated some importance of the 5′initial sequence and a few positions in this sequence are identified bythe codon-position analysis as correlating with expression. One site ofinterest is the codon for serine at position 10 and its neighboringcodons at positions 9 and 11. In only the three top variants (6, 19 AND20), the rather rare codon TCG was used at position 10. Also, strongbiases are observed at positions 3 (CAC>CAT), 9 (TAC>TAT), 10(TCG>TCC>others), 11 (TGC>TGT) and 13 (TTC>TTT).

More than 250 codon-positions had significant positive and negative loadon expression according to the PLS model generated according to theirvariable importance in projection (VIP) scores for the model. VIP is ameasure of the contribution of the variable to the model fitness basedon comparison of models including or singly excluding the variable.Higher VIP means that error in the model is more highly increased uponomission of the variable. These higher VIP variables could be consideredmore likely to be critical for expression. Generally a VIP greater than1 is considered significant to the model.

The importance of so many codon positions for expression implies thatthere is an overall codon bias requirement rather than a small number ofrate-limiting positions or regions. To test this we synthesized 6 newgenes designed as hybrids constructed from variants 19 and 15. Variant19 was chosen because it was the variant expressing the highest levelsof phi29. Variant 15 was chosen because it was the variant with lowestsequence identity to variant 19 that still had detectable levels ofexpressed protein. The parental genes (variants 15 and 19) were dividedinto three segments from codons 1-75, 76-325, and 326-575 and all sixpossible combinations were constructed and tested for expression as forthe original variants (Table A).

All segments were found to be critical and essentially independent intheir contributions to expression. Any substitution of a segment invariant 19 with the corresponding segment from variant 15 reducedexpression 3- to 4-fold. Replacement of the 3′ segment was mostdeleterious. Conversely, substitutions of either 5′ or 3′ segments ofvariant 15 with the corresponding segment from variant 19 increasedexpression 1.5 to 2.5-fold, though we saw no significant improvementwhen the middle segment of variant 15 was replaced. The intermediateexpression levels that result from these hybrid constructs suggest thatthere is not a single dominant rate-determining region (i.e., aparticular cis regulatory element) in the gene coding sequence. Insteadit is consistent with the overall codon bias being the primarydeterminant of the expression levels observed.

TABLE A Relative expression of phi29 polymerase gene variants 15 and 19and hybrids between the two. Parent Parent for for Parent for segmentsegment segment Relative Variant 1-75 76-325 326-575 Expression 15 20 ±4 19 301 ± 65 Hyb 15-15-19 15 15 19  51 ± 17 Hyb 15-19-15 15 19 15 22 ±3 Hyb 19-15-15 19 15 15 30 ± 4 Hyb 15-19-19 15 19 19 101 ± 7  Hyb19-15-19 19 15 19 106 ± 11 Hyb 19-19-15 19 19 15 75 ± 8

The overall codon biases for variants 15 and 19 are quite different fromeach other and from that found in E. coli class II genes, as shown inFIG. 7. The bias in variant 15 is much closer to that of E. coli classII genes than is the bias in variant 19. The codon bias in variant 19 ismuch “flatter” than in variant 15, that is the codons for each aminoacid are much more evenly distributed.

A new partial least squares (PLS) model was constructed using PLSToolbox 4.0 software (Eigenvector, Inc.) within MATLAB (Mathworks, Inc).This model used the 59 codons that had synonymous alternatives (that isall codons except the three stop codons, ATG (Met) and TGG (Trp)) as theindependent variables to describe the polynucleotide sequence property.The latent variables, variable importance in projection and regressionvector in Y were calculated for each codon, the fit between measured andpredicted expression levels are shown in FIG. 8.

A new partial least squares (PLS) model was constructed using theGenetic Algorithm function in PLS Toolbox 4.0 software (Eigenvector,Inc.) within MATLAB (Mathworks, Inc). The genetic algorithm comparedmodels using 100 different random subsets of 15-25 of the 61 sensecodons, and evaluated their ability to explain the data in PLS modeling.The error of the PLS fit of the data in cross validation (RMSECV) wasused to distinguish the subsets. Those that yielded lower than medianRMSECV were retained. The codon sets used by random pairs of theseselected samples were then recombined at two randomly selected crossoverpoints to create new progeny samples. The resulting samples, theoriginal selected and their progeny, were then analyzed for fit asbefore and the best half were used to create the next sample generation.At each generation, mutation (substituting one codon variable in asample for another) was allowed to prevent the model from prematurelyeliminating or fixing under- or overrepresented variables, respectively.The entire process was repeated until there was convergence in makeupand performance of the selected population. In this way we identified 12codons that were most significant for expression. The fit betweenmeasured and predicted expression levels is shown in FIG. 11.

6.2 DESIGN, SYNTHESIS & EXPRESSION TESTING OF A CODON VARIANT SET FOR ASINGLE CHAIN ANTIBODY FRAGMENT 6.2.1 ScFv Variant Design

We created a set of gene variants encoding a 281 codon single-chainantibody fragment (scFv). The scFv gene variants were diversified in asimilar fashion to the phi29 polymerase variants, as described in detailin section 6.1.1.

A set of 6 gene design parameters were varied between the designs. Thesewere:

1. Codon bias. Codon bias describes the relative frequency with whichsynonymous codons are used to encode an amino acid. We used two codonbias tables, one calculated from all of the genes in the E. coli genome(‘E. coli’ table) and a second from a subset of 27 genes that are highlyexpressed (‘E. coli II’ table) (Gustafsson et al., 2004, “Codon bias andheterologous protein expression,” Trends Biotechnol: 22, 346-353; Henautand Danchin, 1996, “Analysis and predictions from Escherichia colisequences,” Escherichia coli and Salmonella typhimurium Cellular andMolecular Biology 2, 2047-2066; Sharp and Li, 1987, “The codonAdaptation Index—a measure of directional synonymous codon usage bias,and its potential applications,” Nucleic Acids Res 15, 1281-1295). Wealso created 3 “flattened” tables based on each of these. This wasachieved by reducing the difference between the frequency of a codon inthe codon bias table used and the mean frequency for each codon encodingthat amino acid. For example, lysine is encoded by AAA 79% of the timeand AAG 21% of the time in E. coli class II genes. The mean of thesefrequencies is 50%. Flattening this codon bias by 50% would reduce thefrequency of AAA by (79−50)/2=14.5% to 64.5%, and increase the frequencyof AAG by (50−21)/2=14.5% to 35.5%. A 100% flattening would result inall possible codons above threshold are used at equal frequency. Thecutoff thresholds used, however, were based on the codon frequencies inthe original codon table. To design each gene variant encoding ScFv, weused Gene Designer software (Villalobos et al, 2006, “Gene Designer: asynthetic biology tool for constructing artificial DNA segments,” BMCBioinformatics: 7, 285). For each consecutive amino acid the programchooses a codon with a probability that is proportional to its frequencyin the chosen bias table. Thus the overall bias of the gene approximatesthat of the bias table.

2. Cutoff threshold. Several previous reports have shown that use ofsome rarely used codons may lower expression, particularly when theseare used frequently or in tandem (Kane, 1995, “Effects of rare codonclusters on high-level expression of heterologous proteins inEscherichia coli,” Curr Opin Biotechnol 6, 494-500; Shu et al., 2006,“Inhibition of translation by consecutive rare leucine codons in E.coli: absence of effect of varying mRNA stability,” Gene Expr: 13,97-106. We therefore used Gene Designer to exclude codons whosefrequencies in the codon bias table are lower than a selected threshold(the cutoff threshold). We varied this threshold to test whetherinclusion of codons used at intermediate frequency or occasionalinclusion of rare codons have a detrimental effect. At the highestthreshold settings used (25%, that is, the only codons allowed are thosethat are used to encode an amino acid more than 25% of the time in thegenes from which that codon bias table is derived), 5-6 amino acids thatcould be encoded by multiple codons are restricted to a single choice.At the lowest settings (2%) only 0-6 of the rarest codons are excluded.

3. Internal RNA structure. After initial coding we analyzed the RNAsecondary structure of each variant using UNAFold (Markham and Zuker,2005, “DINAMelt web server for nucleic acid melting prediction,” NucleicAcids Res 33, W577-W581). RNA structures were determined for every 50nucleotide window of the coding sequence, and for the 3′ terminal tailof the mRNA consisting of the last 50 nucleotides of the coding sequencethrough to the expected terminus. A 50 nucleotide window was usedbecause it should identify local structures that can form betweenconsecutive ribosomes on an mRNA: ribosome spacing is estimated at 50nucleotides for a rapidly translating message. There were multiplepossible strong internal RNA structures but only one, near position 83of the coding sequence, could be engineered mostly independent of codonbias. RNA structure at several positions were analyzed and included inmodeling, but none, including that at 83, contributed significantly tofitting.

4. RNA structure at the 5′ initiation site. We used UNAFold to calculatethe RNA structure from the 5′ end of the mRNA to position +50. Thisstructure was manually modified in selected genes by minimal codonsubstitutions, within the bias and threshold limits.

5. 5′AT wobble. We manually modified the percentage of the first 15codons that use A or T in the 3^(rd) position 6. GC runs. Runs of Gand/or C of 6 or 7 nucleotides or more were avoided in some sequences bydefining SSSSSS/S(S=G or C) as a motif to avoid in Gene Designer.

Five parameters (codon bias (including degree of “flattening”),threshold, internal RNA structure, AT wobble, and GC runs), weresystematically varied. The design properties of the first 24 variantsare shown in Table B.

TABLE B Gene design parameter matrix for scFv test variants. Expressionis relative to variant 13. Codon 5′ ScFv bias % RNA RNA 5′AT Var. tableThreshold Flat^(b) CAI (dG@83) (dG) Wobble GC6 GC7 Expression V1 E. coli0.20 50 0.718 −17.6 −26.9 0.400 7 2 281 ± 27 V2 E. coli 0.05 50 0.443−11.0 −28.6 0.067 7 3 21 ± 6 V3 E. coli II 0.20 50 0.804 −19.6 −26.60.200 11 4  78 ± 11 V4 E. coli II 0.02 50 0.556 −22.5 −27.2 0.667 10 7173 ± 17 V5 E. coli II 0.02 0 0.703 −13.3 −28.0 0.667 9 2 163 ± 17 V6 E.coli II 0.25 0 0.863 −22.8 −26.1 0.067 8 3 152 ± 19 V7 E. coli 0.20 00.716 −24.8 −26.9 0.400 11 6 ND V8 E. coli 0.03 0 0.472 −18.4 −25.00.133 9 4 ND V9 E. coli II 0.20 50 0.825 −16.9 −26.2 0.400 6 0 221 ± 22V10 E. coli II 0.02 50 0.552 −27.2 −27.2 0.200 4 1 109 ± 25 V11 E. coli0.20 50 0.719 −18.0 −27.9 0.067 12 5 ND V12 E. coli 0.05 50 0.395 −23.8−25.9 0.800 10 6 182 ± 28 V13 E. coli 0.02 0 0.459 −12.3 −26.0 0.733 9 1100 ± 12 V14 E. coli 0.20 0 0.742 −27.9 −30.1 0.067 7 2 ND V15 E. coliII 0.27 0 0.858 −23.6 −27.6 0.400 6 5  55 ± 13 V16 E. coli II 0.02 00.714 −17.2 −33.8 0.133 8 4 ND V17 E. coli II 0.10 0 0.815 −17.3 −24.80.400 8 4  61 ± 9 V18 E. coli II 0.10 0 0.759 −19.4 −28.9 0.333 5 3  71± 5 V19 E. coli II 0.10 0 0.8 −18.6 −28.6 0.267 10 4 ND V20 E. coli II0.20 100 0.788 −19.0 −26.7 0.200 7 2  63 ± 10 V21 E. coli II 0.10 1000.665 −17.4 −25.6 0.333 3 0 241 ± 24 V22 E. coli 0.07 100 0.404 −18.4−23.7 0.400 8 2 107 ± 10 V23 E. coli II 0.10 0 0.794 −16.6 −27.5 0.33310 4 182 ± 19 V24 E. coli II 0.10 0 0.797 −21.4 −25.9 0.267 10 6 ND

6.2.2 ScFv Variant synthesis and cloning

The gene variants were all synthesized by standard gene synthesisprocedures and cloned into a pET24a expression vector between the XbaIand EcoRI restriction sites. This put each gene under transcriptionalcontrol of the strong T7 RNA polymerase promoter of this vector. Eachfinal construct was completely sequenced in both directions to becertain that the complete sequence was as designed. The plasmid was thenused to transform E. coli expression host strain BL21(DE3) harboring asecond plasmid for low level expression of T7 lysozyme, pLysS. Thebackground expression of T7 lysozyme, an inhibitor of T7 RNA polymerase,gives tight repression of heterologous expression prior to induction tominimize potential gene toxicity which could affect data quality.

6.2.3 ScFv Variant Expression

Proteins were expressed from the variant genes using standard methods.Prior to analysis of the variants, expression was analyzed for multiplevariants to determine appropriate expression time and temperature.Strong, consistent expression was achieved at 30° C., a commonly usedtemperature for heterologous expression in E. coli. Time courses at 30°C. showed expressed protein levels increasing to a maximum afterapproximately two hours, as the cells entered stationary phase growth,and remaining steady for at least five hours. Relative proteinexpression levels between variants were consistent throughout the timecourse. For our variant analysis we chose to express for four hours at30° C.

For each variant, three transformants were picked and cultured overnightin 2 ml Luria Broth (LB) containing appropriate antibiotics to maintainthe expression vector and pLysS plasmid. The overnight cultures werediluted 50-fold in fresh media and incubated at 37° C. until the cellswere in mid-log growth (OD at 600 nm˜0.6). Expression was induced byaddition of IPTG to 1 mM and incubation for four hours at 30° C. Finaloptical densities of cultures were measured and equivalent amounts ofculture were analyzed by polyacrylamide gel electrophoresis (a sampleset of variant expression is shown in FIG. 9). Gels were stained withSypro Ruby (Pierce), visualized by fluorescence imaging, and proteinband intensities quantified using TotalLab 100 image analysis software(Nonlinear Dynamics, Inc). Each gel contained protein concentrationstandards to calibrate band intensity. Three replicates of a referenceclone (ScFv variant 13) were expressed in parallel cultures in eachexperiment to allow corrections to be made for any experiment toexperiment variation. Reported expression levels are all relative tothis reference. The detection limit of the assay was approximately 1 μgof ScFv per ml culture at an A₆₀₀=3. The standard error of measuredexpression for variant repeats was generally <20% of the mean.

Quantitation of expression levels for the variants was assessed. Thereare a wide range of expression levels among the variants, demonstratinga good sampling of expression relevant features in our gene designs.Levels ranged from below detection to three times the reference, a rangeof two orders of magnitude.

6.2.4 Analysis of Gene Design Parameters for ScFv Variants

To determine the influence of the various gene design parameters onexpression, the data was fit to partial least squares (PLS) models usingPLS Toolbox 4.0 software (Eigenvector, Inc.) within MATLAB (Mathworks,Inc). PLS regression is a highly reliable method for modeling systemsdata where the number of independent variables is high, approaching oreven exceeding the number of samples (Eriksson et al., Wold, 2004,“Using chemometrics for navigating in the large data sets of genomics,proteomics, and metabonomics (gpm),” Anal Bioanal Chem: 380, 419-429).In PLS regression, multivariate data are transformed to a variable spacewith reduced dimensionality that is defined by new orthogonal variables(latent variables). The latent variables are linear combinations of theoriginal variables, calculated to maximize correlation betweenindependent and dependent variables in as few dimensions as possible.Cross-validation methods are used to determine the optimal number oflatent variables to use in the model, so that fit between data and modelis maximized without over-fitting.

Correlation between observed and predicted expression levels for aninitial PLS model of the ScFv variants data is shown in FIG. 10 part A.FIG. 10 part B shows the contributions of varied parameters to theexpression of the scFv. Most striking, and consistent with ourpredictions from phi29 variants in Example 6.1, we observe a positiveeffect of flattening the codon bias. The three highest expressingvariants all have flattened bias, including the 100% flattened variant21. These results indicate that there is an advantage to sampling a morediverse codon usage than is typically done in standard geneoptimization. Also consistent with our results from phi29 polymerase,CAI does not correlate positively with expression. In fact, once again,we observe a negative effect of high CAI and, more weakly, threshold,again suggesting that a different codon bias is optimal.

We also see correlation of expression with 5′ codon usage. Both 5′-ATWobble and 5′-RNA structure correlate with expression, although in thiscase higher A/T usage and weaker structure is preferred, opposite of thepreferences seen with phi29 polymerase. As with the phi29 polymerase, ATwobble and 5′ RNA structure are heavily influenced by codon biases.

A new partial least squares (PLS) model was constructed using PLSToolbox 4.0 software (Eigenvector, Inc.) within MATLAB (Mathworks, Inc).This model used the 59 codons that had synonymous alternatives (that isall codons except the three stop codons, ATG (Met) and TGG (Trp) as theindependent variables to describe the polynucleotide sequence property.The latent variables, variable importance in projection and regressionvector in Y were calculated for each codon, the fit between measured andpredicted expression levels for this model are shown in FIG. 12.

A new partial least squares (PLS) model was constructed using theGenetic Algorithm function in PLS Toolbox 4.0 software (Eigenvector,Inc.) within MATLAB (Mathworks, Inc). The genetic algorithm comparedmodels using 100 different random subsets of 15-25 of the 61 sensecodons, and evaluated their ability to explain the data in PLS modeling.The error of the PLS fit of the data in cross validation (RMSECV) wasused to distinguish the subsets. Those that yielded lower than medianRMSECV were retained. The codon sets used by random pairs of theseselected samples were then recombined at two randomly selected crossoverpoints to create new progeny samples. The resulting samples, theoriginal selected and their progeny, were then analyzed for fit asbefore and the best half were used to create the next sample generation.At each generation, mutation (substituting one codon variable in asample for another) was allowed to prevent the model from prematurelyeliminating or fixing under- or overrepresented variables, respectively.The entire process was repeated until there was convergence in makeupand performance of the selected population. In this way we identified 12codons that were most significant for expression. The fit betweenmeasured and predicted expression levels is shown in FIG. 13.

6.3 COMBINED SEQUENCE-EXPRESSION ANALYSIS OF CODON VARIANT SETS FOR PHI29 DNA POLYMERASE AND A SINGLE CHAIN ANTIBODY FRAGMENT 6.3.1 ModelingCodon Expression Data

A partial least squares (PLS) model was constructed for the combineddata for phi29 and ScFv variants for which activity data was shown inFIG. 2. The model was constructed using PLS Toolbox 4.0 software(Eigenvector, Inc.) within MATLAB (Mathworks, Inc). This model used the59 codons that had synonymous alternatives (that is all codons exceptthe three stop codons, ATG (Met) and TGG (Trp)) as the independentvariables to describe the polynucleotide sequence property. The latentvariables, variable importance in projection and regression vector in Ywere calculated for each codon. The fit between measured and predictedexpression levels for this model are shown in FIG. 14.

A new partial least squares (PLS) model was constructed for a datasetincluding both the ScFv and phi29 sequences using the Genetic Algorithmfunction in PLS Toolbox 4.0 software (Eigenvector, Inc.) within MATLAB(Mathworks, Inc). The genetic algorithm compared models using 100different random subsets of 15-25 of the 61 sense codons, and evaluatedtheir ability to explain the data in PLS modeling. The error of the PLSfit of the data in cross validation (RMSECV) was used to distinguish thesubsets. Those that yielded lower than median RMSECV were retained. Thecodon sets used by random pairs of these selected samples were thenrecombined at two randomly selected crossover points to create newprogeny samples. The resulting samples, the original selected and theirprogeny, were then analyzed for fit as before and the best half wereused to create the next sample generation. At each generation, mutation(substituting one codon variable in a sample for another) was allowed toprevent the model from prematurely eliminating or fixing under- oroverrepresented variables, respectively. The entire process was repeateduntil there was convergence in makeup and performance of the selectedpopulation. In this way we identified 16 codons that were mostsignificant for expression. The fit between measured and predictedexpression levels is shown in FIG. 15.

6.3.2 Design of a New Codon Bias Table

A new codon bias was calculated by calculating the average usage of eachcodon, in terms of codon frequency per codon position, in the top 10expressed clones for the two gene variant sets. Thus a table was createdto approximate the codon usage of highly expressed clones discoveredthrough systematic gene variation and analysis.

6.4 APPLICATION OF CODON BIAS MODEL TO CENPA EXPRESSION IN E. COLI

Initial attempts to express the Xenopus laevis cenpA protein in E. coliusing natural and re-designed synthetic genes had yielded no detectableproduct. In an attempt to increase yield, a new gene design was createdbased on our initial modeling results. A codon table, “ModA,” wasconstructed in which the frequency usage of codons was weighted by thevariable loadings in a model constructed from the combined scFv andphi29 DNA polymerase datasets as described above. For each amino acid,codons used at frequencies of less than 5% in E. coli genes wereexcluded from the table (set to zero frequency). The ModA table was thenapplied to cenpA gene design. Another design, where the codonfrequencies were matched to those found in 27 highly expressed E. coligenes (Table “Coli-II”) was also created. All design parameters otherthan codon frequency usage were similar for the two genes. The geneswere expressed under identical conditions in identical E. coli hoststrains and expression level was analyzed by PAGE (FIG. 16). Nosignificant expression relative to control strains (lacking cenpA) wasobserved from the Coli-II derived gene whereas significantly moreproduct was seen from the ModA gene.

6.5 SEQUENCES

SEQAA (5′Constant Vector Sequence)

TGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAATTAATTCTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTAGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCTGCGGTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACAGATGTCTGCCTGTTCATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAAGCGGGCCATGTTAAGGGCGGTTTTTTCCTGTTTGGTCACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCATGGGGGTAATGATACCGATGAAACGAGAGAGGATGCTCACGATACGGGTTACTGATGATGAACATGCCCGGTTACTGGAACGTTGTGAGGGTAAACAACTGGCGGTATGGATGCGGCGGGACCAGAGAAAAATCACTCAGGGTCAATGCCAGCGCTTCGTTAATACAGATGTAGGTGTTCCACAGGGTAGCCAGCAGCATCCTGCGATGCAGATCCGGAACATAATGGTGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTACGAAACACGGAAACCGAAGACCATTCATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCATTCTGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCAACGACAGGAGCACGATCATGCGCACCCGTGGGGCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACGATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGATCCCGCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATTACC (SEQ ID NO: 1)

SEQAB (3′Constant Vector Sequence)

TGATAAAGTGGCTCCAACACTTCCTACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGAT (SEQ IDNO: 2)

gtgggttctg aaagtgcagg cggatctgta ctttcacaac ctgaaattcg (SEQ ID NO: 3)

ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaaggagatattac catgaagcac atgccgcgga agatgtactc ctgcgacttc gagacgacca c (SEQID NO: 4)

7. REFERENCES CITED

All references cited herein are incorporated herein by reference intheir entirety and for all purposes to the same extent as if eachindividual publication or patent or patent application was specificallyand individually indicated to be incorporated by reference in itsentirety herein for all purposes.

8. MODIFICATIONS

Many modifications and variations of this invention can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. The specific embodiments described herein areoffered by way of example only, and the invention is to be limited onlyby the terms of the appended claims, along with the full scope ofequivalents to which such claims are entitled.

1. A method of determining at least one property that affects anexpression property value of polynucleotides in an expression system,the method comprising: (A) constructing a plurality of polynucleotides,wherein the plurality of polynucleotides comprises five or morepolynucleotides, each respective polynucleotide in the five or morepolynucleotides encoding a respective single polypeptide sequence theentirety of which is at least ninety-five percent identical to theentirety of the respective single polypeptide sequence encoded by eachother polynucleotide in the five or more polynucleotides, wherein (i) afirst amino acid is encoded a first plurality of times in a firstpolynucleotide, a second polynucleotide, and a third polynucleotide inthe five or more polynucleotides, (ii) the first amino acid is encodableby a plurality of synonymous codons including a first codon, (iii) thefirst codon is present in the first polynucleotide with a firstfrequency relative to all other codons in the plurality of synonymouscodons in the first polynucleotide, (iv) the first codon is present inthe second polynucleotide with a second frequency relative to all othercodons in the plurality of synonymous codons in the secondpolynucleotide, (v) the first codon is present in the thirdpolynucleotide with a third frequency relative to all other codons inthe plurality of synonymous codons in the third polynucleotide; and (vi)the first frequency is different than the second frequency and the thirdfrequency is between the first frequency and the second frequency; (B)expressing each respective polynucleotide in the plurality ofpolynucleotides individually in said expression system using the samevector and the same promoter; (C) measuring an expression property valueof each respective polynucleotide in the plurality of polynucleotides inthe expression system; and (D) determining at least one property thataffects an expression property value of polynucleotides in theexpression system, wherein the at least one property is an effect that afrequency of use of (i) the first codon or (ii) the first codon and oneor more other codons in a plurality of naturally occurring codons has onthe protein expression property values of polynucleotides in theexpression system; and the expression property value of each respectivepolynucleotide in the plurality of polynucleotides in the expressionsystem is (i) a total amount of protein encoded by the respectivepolynucleotide that is expressed in the expression system in apredetermined period of time (ii) a total amount of active proteinencoded by the respective polynucleotide that is expressed in theexpression system in a predetermined period of time, or (iii) a totalamount of soluble protein encoded by the respective polynucleotide thatis expressed in the expression system in a predetermined period of time.2. The method of claim 1, wherein the expression property value of eachrespective polynucleotide in the plurality of polynucleotides in theexpression system is a total amount of protein encoded by the respectivepolynucleotide that is expressed in the expression system in apredetermined period of time.
 3. The method of claim 1, wherein theexpression property value of each respective polynucleotide in theexpression system is a total amount of active protein encoded by therespective polynucleotide that is expressed in the expression system ina predetermined period of time.
 4. The method of claim 1, wherein theexpression property value of each respective polynucleotide in theexpression system is a total amount of soluble protein encoded by therespective polynucleotide that is expressed in the expression system ina predetermined period of time.
 5. The method of claim 1 wherein, foreach respective amino acid in a plurality of amino acids comprising fiveor more amino acids, a relative frequency of each of a plurality ofsynonymous codons for the respective amino acid is varied in a region ofeach of two or more of the polynucleotides in the plurality ofpolynucleotides.
 6. The method of claim 1 wherein, for each respectiveamino acid in a plurality of amino acids comprising two or more aminoacids, a relative frequency of each of a plurality of synonymous codonsfor the respective amino acid is varied in a region of each of five ormore of the polynucleotides in the plurality of polynucleotides.
 7. Themethod of claim 1, wherein the plurality of polynucleotides comprisesten or more polynucleotides.
 8. The method of claim 1, wherein theplurality of polynucleotides comprises twenty or more polynucleotides.9. The method of claim 1, wherein the constructing (A) comprises:encoding the first polynucleotide using a first frequency lookup table,wherein the first frequency lookup table specifies a first targetfrequency or target frequency range for the use of the first codonrelative to all other codons in the plurality of synonymous codons in apolynucleotide and wherein the first frequency of (A)(iii) is within thefirst target frequency or target frequency range; and encoding thesecond polynucleotide using a second frequency lookup table, wherein thesecond frequency lookup table specifies a second target frequency ortarget frequency range for the use of the first codon relative to allother codons in the plurality of synonymous codons in a polynucleotideand wherein the second frequency of (A)(iv) is within the second targetfrequency or target frequency range; and wherein the first targetfrequency or target frequency range is different from the second targetfrequency or target frequency range.
 10. The method of claim 9, whereinthe plurality of synonymous codons is three or more different codons.11. The method of claim 9, wherein a third frequency lookup tablespecifies a third target frequency or target frequency range for the useof the first codon relative to all other codons in the plurality ofsynonymous codons in the third polynucleotide in the plurality ofpolynucleotides.
 12. The method of claim 1, wherein a first frequencylookup table specifies a corresponding respective target frequency orfrequency range for each codon in a first plurality of codons, eachcorresponding respective target frequency or target frequency rangespecifying a target frequency or target frequency range for a codon inthe first plurality of codons to be used to encode a corresponding aminoacid in an amino acid sequence relative to all other codons that arecapable of encoding the corresponding amino acid, and wherein for eachrespective codon in the first frequency lookup table, the constructing(A) further comprises choosing a respective frequency that therespective codon is to be used to encode the amino acid encodable by therespective codon throughout an amino acid sequence encoded by said firstpolynucleotide in the plurality of polynucleotides relative to all othercodons capable of encoding the amino acid, wherein the respectivefrequency is the frequency or is within the frequency range specified inthe first frequency lookup table for the respective codon; and a secondfrequency lookup table specifies a corresponding respective targetfrequency or frequency range for each codon in a second plurality ofcodons, each corresponding respective target frequency or targetfrequency range specifying a target frequency or target frequency rangefor a codon in the second plurality of codons to be used to encode acorresponding amino acid in an amino acid sequence relative to all othercodons that are capable of encoding the corresponding amino acid, andwherein for each respective codon in the second frequency lookup table,the constructing (A) further comprises choosing a respective frequencythat the respective codon is to be used to encode the amino acidencodable by the respective codon throughout an amino acid sequenceencoded by said second polynucleotide in the plurality ofpolynucleotides relative to all other codons capable of encoding theamino acid, wherein the respective frequency is within the frequency orthe frequency range specified in the second frequency lookup table forthe respective codon; wherein the first frequency of (A)(iii) is afrequency or is within a frequency range specified for the first aminoacid by the first frequency lookup table; and the second frequency of(A)(iv) is a frequency or is within a frequency range specified for thefirst amino acid by the second frequency lookup table.
 13. The method ofclaim 1, wherein the constructing (A) comprises: encoding a first set ofpositions in the first polynucleotide using a first frequency lookuptable, wherein the first frequency lookup table specifies a first targetfrequency or frequency range for the use of a predetermined codon,relative to all other codons that are synonymous to the predeterminedcodon, in the first set of positions in the first polynucleotide thatencode a predetermined amino acid; and encoding a second set ofpositions in the first polynucleotide using a second frequency lookuptable, wherein the second frequency lookup table specifies a secondtarget frequency or frequency range for the use of the predeterminedcodon, relative to all other codons that are synonymous to thepredetermined codon, in the second set of positions in the firstpolynucleotide that encode the predetermined amino acid; wherein thefirst set of positions do not include positions in the second set ofpositions; and the second set of positions do not include positions inthe first set of positions.
 14. The method of claim 13, wherein thepredetermined amino acid can be encoded by three or more synonymouscodons in the plurality of synonymous codons.
 15. The method of claim 1,wherein a first frequency lookup table specifies a correspondingrespective first target frequency or frequency range for each codon in afirst plurality of codons, each corresponding respective first targetfrequency or frequency range specifying a first target frequency orfrequency range for a codon in the first plurality of codons to be usedto encode a corresponding amino acid in an amino acid sequence relativeto all other codons that are capable of encoding the corresponding aminoacid, and wherein for each respective codon in the first frequencylookup table, the constructing (A) further comprises choosing arespective first frequency that the respective codon is to be used toencode the amino acid encodable by the respective codon in apredetermined first set of positions in the amino acid sequence encodedby said first polynucleotide in the plurality of polynucleotidesrelative to all other codons capable of encoding the amino acid, whereinthe respective first frequency is within the first target frequency orfrequency range specified in the first frequency lookup table for therespective codon; and a second frequency lookup table specifies acorresponding respective second target frequency or frequency range foreach codon in a second plurality of codons, each correspondingrespective second target frequency or frequency range specifying asecond target frequency or frequency range for a codon in the secondplurality of codons to be used to encode a corresponding amino acid inan amino acid sequence relative to all other codons that are capable ofencoding the corresponding amino acid, and wherein for each respectivecodon in the second frequency lookup table, the constructing (A) furthercomprises choosing a respective second frequency that the respectivecodon is to be used to encode the amino acid encodable by the respectivecodon in a predetermined second set of positions in the amino acidsequence encoded by said first polynucleotide in the plurality ofpolynucleotides relative to all other codons capable of encoding theamino acid, wherein the respective second frequency is within the secondtarget frequency or frequency range specified in the second frequencylookup table for the respective codon.
 16. The method of claim 1,wherein the expression system is E. coli, baculovirus, a mammaliantissue culture, yeast, or a plant.
 17. The method of claim 1, whereinthe first frequency the second frequency and the third frequency areselected using an experimental design technique.
 18. The method of claim1 wherein, the first frequency, the second frequency and the thirdfrequency are selected so that they fit a multivariate space that can besearched using a global optimization algorithm.
 19. A method ofdetermining at least one property that affects an expression propertyvalue of polynucleotides in an expression system, the method comprising:(A) defining a predetermined frequency or frequency range forincorporation of a first codon into a polynucleotide wherein thedefining comprises defining a first frequency, a second frequency, and athird frequency for the first codon based upon the predeterminedfrequency or frequency range; (B) constructing a plurality ofpolynucleotides, wherein the plurality of polynucleotides comprises fiveor more polynucleotides, each respective polynucleotide in the five ormore polynucleotides encoding a respective single polypeptide sequencethe entirety of which is at least ninety-five percent identical to theentirety of the respective single polypeptide sequence encoded by eachother polynucleotide in the five or more polynucleotides wherein (i) afirst amino acid is encoded a first plurality of times in a firstpolynucleotide, a second polynucleotide, and a third polynucleotide inthe five or more polynucleotides, (ii) the first amino acid is encodableby a plurality of synonymous codons including the first codon, (iii) thefirst codon is present in the first polynucleotide with the firstfrequency, (iv) the first codon is present in the second polynucleotidewith the second frequency, (v) the first codon is present in the thirdpolynucleotide with the third frequency, and (vi) the first frequency isdifferent than the second frequency, and the third frequency is betweenthe first frequency and the second frequency, and (C) expressing eachrespective polynucleotide in the plurality of polynucleotidesindividually in said expression system using the same vector and thesame promoter; and (D) measuring an expression property value of eachrespective polynucleotide in the plurality of polynucleotides in theexpression system thereby determining at least one property that affectsan expression property value of polynucleotides in the expressionsystem, wherein the at least one property is an effect that a frequencyof use of one or more codons in a plurality of naturally occurringcodons has on the expression property values of polynucleotides in theexpression system; the expression property value of each respectivepolynucleotide in the plurality of polynucleotides in the expressionsystem is (i) a total amount of protein encoded by the respectivepolynucleotide that is expressed in the expression system in apredetermined period of time (ii) a total amount of active proteinencoded by the respective polynucleotide that is expressed in theexpression system in a predetermined period of time, or (iii) a totalamount of soluble protein encoded by the respective polynucleotide thatis expressed in the expression system in a predetermined period of time.20. The method of claim 19, wherein the first frequency, the secondfrequency and the third frequency are selected probabilistically basedupon the predetermined frequency or frequency range.